preparation-pancreatic-acinar-cells-for-purpose-calcium-imaging-cell

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Children's Hospital of Pittsburgh of UPMC

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.

Pancr&eacute;atite aigu&euml; est une maladie douloureuse, inflammatoire pour lesquels les traitements ad&eacute;quats font d&eacute;faut. Une &eacute;tape pr&eacute;coce, critique de son d&eacute;veloppement est la signalisation aberrante de Ca(2+) dans les cellules acineuses du pancr&eacute;as. Cette Ca(2+) de sortie est modul&eacute; par le Ca(2+) intracellulaire canal du r&eacute;cepteur de la ryanodine (RYR). Nous avons d&eacute;j&agrave; montr&eacute; que l&#39;inhibition RYR r&eacute;duit activation de prot&eacute;ases intra-acinar pathologique, un marqueur pr&eacute;coce de la pancr&eacute;atite. Dans cette &eacute;tude, nous avons examin&eacute; si un pr&eacute;traitement avec le dantrol&egrave;ne inhibiteur RYR att&eacute;nue la gravit&eacute; de la pancr&eacute;atite chez les souris induite par le c&eacute;rul&eacute;ine. L&#39;&eacute;tiquetage par immunofluorescence &agrave; Adieu bazou de sections du pancr&eacute;as de souris ont montr&eacute; la localisation dans la r&eacute;gion basolat&eacute;rale de la cellule acineuse. Apr&egrave;s 1 h d&#39;une hyperstimulation c&eacute;rul&eacute;ine in vivo, dantrol&egrave;ne 1) r&eacute;duit l&#39;activit&eacute; trypsine pancr&eacute;atique de 59 % (P 0,05 &lt;) et 2) att&eacute;nu&eacute; les d&eacute;rangements ultrastructurales pr&eacute;coces au sein de la cellule acineuse. Huit heures apr&egrave;s l&#39;induction de la pancr&eacute;atite, dantrol&egrave;ne r&eacute;duit l&#39;activit&eacute; trypsine pancr&eacute;atique et amylase s&eacute;rique de 61 % et 32 % respectivement (P 0,05 &lt;). &Agrave; ce moment plus tard, gravit&eacute; globale histologique de pancr&eacute;atite a &eacute;t&eacute; r&eacute;duite de 63 % avec un pr&eacute;traitement dantrol&egrave;ne (P 0,05 &lt;). Cellules TUNEL-positives ont &eacute;t&eacute; r&eacute;duits de 58 % (P 0,05 &lt;). Ces r&eacute;sultats sugg&egrave;rent que le RYR joue un r&ocirc;le important dans la m&eacute;diation des &eacute;v&eacute;nements pr&eacute;coces de cellules acineuses au cours de la pancr&eacute;atite in vivo et qu&#39;il contribue &agrave; la gravit&eacute; de la maladie. Blocus de Ca(2+) des signaux et en particulier de RYR-Ca(2+) peut &ecirc;tre utile comme traitement prophylactique pour cette maladie dans des milieux &agrave; risque &eacute;lev&eacute; pour la pancr&eacute;atite.

Dantrol&egrave;ne att&eacute;nue la pancr&eacute;atite induite par le c&eacute;rul&eacute;ine in vivo chez la souris.

Pancreatite acuta &egrave; una malattia infiammatoria, dolorosa per i quali sono privi di trattamenti adeguati. Un passaggio precoce, fondamentale nel suo sviluppo &egrave; la segnalazione aberrante di Ca(2+) all&#39;interno delle cellule acinose del pancreas. Questo Ca(2+) rilascio &egrave; modulata dalla Ca(2+) intracellulari del canale del recettore rianodinico (RYR). In precedenza abbiamo dimostrato che l&#39;inibizione RYR riduce l&#39;attivazione proteasi intra-acinar patologica, un marker precoce di pancreatite. In questo studio, abbiamo esaminato se il pretrattamento con il dantrolene inibitore RYR attenua la gravit&agrave; della pancreatite indotta da caerulein nei topi. Etichettatura immunofluorescenza per RYR da sezioni del pancreas del mouse ha mostrato la localizzazione nella regione basolaterale della cellula acinose. Dopo 1 h di iperstimolazione caerulein in vivo, dantrolene 1) ridotta attivit&agrave; della tripsina pancreatica del 59% (P &lt; 0,05) e 2) mitigata primi derangements ultrastrutturale all&#39;interno delle cellule acinose. Otto ore dopo l&#39;induzione di pancreatite, dantrolene ridotta attivit&agrave; della tripsina pancreatica e amilasi siero da 61 e il 32%, rispettivamente (P &lt; 0,05). In questo secondo tempo, gravit&agrave; complessiva istologica di pancreatite &egrave; stata ridotta del 63% con dantrolene pretrattamento (P &lt; 0,05). Cellule TUNEL-positive sono state ridotte del 58% (P &lt; 0,05). Questi dati suggeriscono che il RYR gioca un ruolo importante nella mediazione i primi eventi di cellule acinose durante la pancreatite in vivo e contribuisce alla gravit&agrave; della malattia. Segnali di blocco del Ca(2+) e particolarmente RYR-Ca(2+) pu&ograve; essere utile come trattamento profilattico per questa malattia in ambienti ad alto rischio per la pancreatite.

Dantrolene mitiga la pancreatite indotta da caerulein in vivo nei topi.

Acute pancreatitis is a necroinflammatory disease that leads to 210,000 hospitalizations in the United States annually. Recent reports suggest that there may be important differences in clinical features between infants/toddlers and older children. Thus, in this study we make a direct comparison between the pediatric age groups in presentation and management trends of acute pancreatitis.

A comparison of presentation and management trends in acute pancreatitis between infants/toddlers and older children.

La pancr&eacute;atite aigu&euml; est une maladie n&eacute;crotico-inflammatoire qui m&egrave;ne &agrave; 210 000 hospitalisations aux Etats-Unis chaque ann&eacute;e. Des rapports r&eacute;cents sugg&egrave;rent qu&#39;il peut y avoir des diff&eacute;rences importantes dans les caract&eacute;ristiques cliniques entre les b&eacute;b&eacute;s/enfants en bas &acirc;ge et les enfants plus &acirc;g&eacute;s. Ainsi, dans cette &eacute;tude, nous faisons une comparaison directe entre l&#39;age groups p&eacute;diatrique dans la pr&eacute;sentation et les tendances de la gestion de la pancr&eacute;atite aigu&euml;.

Une comparaison des tendances de pr&eacute;sentation et de gestion dans la pancr&eacute;atite aigu&euml; entre les b&eacute;b&eacute;s/enfants en bas &acirc;ge et les enfants plus &acirc;g&eacute;s.

Pancreatite acuta &egrave; una malattia necro-infiammatoria che porta a 210.000 ricoveri negli Stati Uniti ogni anno. Rapporti recenti suggeriscono che ci possono essere differenze importanti nelle caratteristiche cliniche tra i neonati/bambini piccoli e bambini pi&ugrave; grandi. Cos&igrave;, in questo studio, facciamo un confronto diretto tra la age groups pediatrica nella presentazione e gestione tendenze di pancreatite acuta.

Un confronto delle tendenze di presentazione e gestione della pancreatite acuta tra i neonati/bambini piccoli e bambini pi&ugrave; grandi.

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

L&#39;activation pr&eacute;matur&eacute;e des proenzymes digestifs, plus pr&eacute;cis&eacute;ment des prot&eacute;ases, dans les cellules acineuses du pancr&eacute;as est un &eacute;v&eacute;nement pr&eacute;coce et critique au cours de la pancr&eacute;atite aigu&euml;. Nos &eacute;tudes pr&eacute;c&eacute;dentes montrent que cette activation n&eacute;cessite une augmentation pathologique dans Ca(2+) cytosolique. En outre, nous avons montr&eacute; qu&#39;une cible de Ca(2+) aberrant dans les cellules acineuses est la calcineurine phosphatase de Ca(2+)/calmoduline-d&eacute;pendante (PP2B). Dans cette &eacute;tude, nous avons &eacute;mis l&#39;hypoth&egrave;se que PP2B intervient dans la s&eacute;v&eacute;rit&eacute; de l&#39;activation et la pancr&eacute;atite prot&eacute;ase in vivo. Pour tester ceci, pancr&eacute;atite a &eacute;t&eacute; induite chez les souris sur 8 h en administrant des injections intrap&eacute;riton&eacute;ales horaires de la chol&eacute;cystokinine analogique c&eacute;rul&eacute;ine (50 microg/kg). Traitement avec l&#39;inhibiteur PP2B FK506, 1 et 8 h apr&egrave;s l&#39;induction de la pancr&eacute;atite r&eacute;duit les activit&eacute;s de la trypsine par plus de 50 % (P &lt; 0,005). IL-6 et l&#39;amylase s&eacute;rique a &eacute;t&eacute; r&eacute;duit de 86 et 84 % par rapport &agrave; la ligne de base (P &lt; 0,0005) &agrave; 8 h, respectivement. Gravit&eacute; histologique de pancr&eacute;atite, class&eacute;e sur la base d&#39;un oed&egrave;me du pancr&eacute;as, une vacuolisation des cellules acineuses, l&#39;inflammation et l&#39;apoptose, a &eacute;t&eacute; r&eacute;duite au d&eacute;but, au cours de la pancr&eacute;atite. Activit&eacute; my&eacute;loperoxydase du pancr&eacute;as et du poumon a &eacute;t&eacute; r&eacute;duite respectivement de 93 et 83 % par rapport &agrave; la ligne de base, (P 0,05 &lt;). Ces donn&eacute;es sugg&egrave;rent que PP2B est une cible importante de la cellule acineuse aberrante Ca(2+) &eacute;l&eacute;vation associ&eacute;e &agrave; la pancr&eacute;atite et l&#39;activation de la prot&eacute;ase pathologique.

Activation de la prot&eacute;ase au cours de la pancr&eacute;atite in vivo d&eacute;pend de l&#39;activation de la calcineurine.

L&#39;attivazione precoce di proenzymes digestivi, in particolare le proteasi, all&#39;interno delle cellule acinose del pancreas &egrave; un evento precoce e critico durante la pancreatite acuta. I nostri precedenti studi dimostrano che questa attivazione richiede un aumento patologico distinto in Ca(2+) citosolico. Inoltre, abbiamo dimostrato che una destinazione di aberrante Ca(2+) nelle cellule acinose &egrave; il Ca(2+)/calmodulina-dipendente fosfatasi calcineurina (PP2B). In questo studio, abbiamo ipotizzato che la PP2B media gravit&agrave; attivazione e pancreatite proteasi in vivo. Per fare una prova, pancreatite &egrave; stata indotta nei topi oltre 8 h dalla somministrazione orarie iniezioni intraperitoneali della colecistochinina analogico caerulein (50 microg/kg). Il trattamento con l&#39;inibitore di PP2B FK506 a 1 e 8 ore dopo l&#39;induzione di pancreatite ridotta attivit&agrave; della tripsina di superiore al 50% (P &lt; 0,005). Amilasi sierica e IL-6 &egrave; stato ridotto da 86 e 84% rispetto al basale (P &lt; 0,0005) a 8 h, rispettivamente. Gravit&agrave; istologica di pancreatite, classificata sulla base di edema del pancreas, vacuolizzazione delle cellule acinose, infiammazione e apoptosi, &egrave; stata ridotta precocemente nel corso della pancreatite. Attivit&agrave; di mieloperossidasi da polmone e pancreas &egrave; stato ridotto da 93 e 83% rispetto al basale, rispettivamente (P &lt; 0,05). Questi dati suggeriscono che la PP2B &egrave; un obiettivo importante della cellula aberrante acinose Ca(2+) aumento associato con pancreatite e attivazione di proteasi patologica.

Attivazione di proteasi durante la pancreatite in vivo &egrave; dipendente dall&#39;attivazione della calcineurina.

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70-90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 Î¼M). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca(2+) wave from 9 to 18 Î¼m/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) wave speed was also observed with a change from physiologic concentrations of carbachol (1 Î¼M) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10-16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca(2+) waves.

Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini.

L&#39;abus d&#39;alcool est des principales causes de pancr&eacute;atite, repr&eacute;sentant 30 % des cas aigus et 70-90 % des cas chroniques, mais les m&eacute;canismes qui conduisent aux l&eacute;sions pancr&eacute;atiques associ&eacute;es &agrave; l&#39;alcool ne sont pas claires. Une fonctionnalit&eacute; critique et d&eacute;but de pancr&eacute;atite est la signalisation aberrante de Ca(2+) dans les cellules acineuses du pancr&eacute;as. Un important chef d&#39;orchestre de cette Ca(2+) est la peut localis&eacute;e, r&eacute;cepteur de la ryanodine Ca(2+) canal intracellulaire (RYR). Dans cette &eacute;tude, nous avons examin&eacute; l&#39;effet d&#39;&eacute;thanol sur la m&eacute;diation tant activation de la prot&eacute;ase intra-acinar pathologique, un pr&eacute;curseur de la pancr&eacute;atite, ainsi que les signaux de RYR Ca(2+). Nous avons &eacute;mis l&#39;hypoth&egrave;se que l&#39;&eacute;thanol sensibilise les cellules acineuses &agrave; l&#39;activation de la prot&eacute;ase en modulant RYR Ca(2+). Les cellules acineuses sont fra&icirc;chement isol&eacute;s de rat, pr&eacute;trait&eacute;s avec de l&#39;&eacute;thanol et stimul&eacute;s par le carbachol agoniste muscarinique (1 &mu;M). L&#39;&eacute;thanol a provoqu&eacute; un doublement dans l&#39;activation induite par le carbachol des prot&eacute;ases trypsine et la chymotrypsine (p &lt; 0,02). Le dantrol&egrave;ne inhibiteur RYR a abrog&eacute; l&#39;am&eacute;lioration de l&#39;activit&eacute; de la trypsine et la chymotrypsine par l&#39;&eacute;thanol (p &lt; 0,005 pour les deux prot&eacute;ases). De plus, l&#39;&eacute;thanol acc&eacute;l&eacute;r&eacute; la vitesse de l&#39;apicale basolat&eacute;rale Ca(2+) vague du 9 au 18 &mu;m/s (p &lt; 0,0005; n = 18-22/groupe de cellules) ; On observe &eacute;galement une augmentation de vitesse de vague de Ca(2+) avec un changement de concentrations physiologiques de carbachol (1 &mu;M) &agrave; une concentration de supraphysiologique (1 mM) qui conduit &agrave; l&#39;activation de la prot&eacute;ase. Dantrol&egrave;ne abrog&eacute; l&#39;acc&eacute;l&eacute;ration induite par l&#39;&eacute;thanol de la vitesse de l&#39;onde (p &lt; 0,05; n = cellules/groupe de 10-16). Nos r&eacute;sultats sugg&egrave;rent que l&#39;am&eacute;lioration de l&#39;activation de la prot&eacute;ase pathologique par l&#39;&eacute;thanol d&eacute;pend de l&#39;Adieu bazou et qu&#39;un nouveau m&eacute;canisme pour que cette am&eacute;lioration peut impliquer acc&eacute;l&eacute;ration RYR m&eacute;di&eacute;e par des ondes de Ca(2+).

&Eacute;thanol am&eacute;liore l&#39;activation de la prot&eacute;ase induite par le carbachol et acc&eacute;l&egrave;re les ondes de Ca2 + dans les acini pancr&eacute;atiques isol&eacute; du rat.

L&#39;abuso di alcol &egrave; delle principali cause di pancreatite, pari al 30% dei casi acuti e 70-90% dei casi cronici, ancora non sono chiari i meccanismi che portano al danno pancreatico associate all&#39;alcol. Una caratteristica precoce e critica di pancreatite &egrave; la segnalazione aberrante di Ca(2+) all&#39;interno delle cellule acinose del pancreas. Un importante direttore d&#39;orchestra di questa Ca(2+) &egrave; la basolaterally localizzata, recettore intracellulare Ca(2+) canale rianodinico del (RYR). In questo studio, abbiamo esaminato l&#39;effetto dell&#39;etanolo su entrambi attivazione proteasi intra-acinar patologica, un precursore di pancreatite, come pure RYR Ca(2+) segnali di mediazione. Abbiamo ipotizzato che etanolo sensibilizza la cellule acinose all&#39;attivazione di proteasi modulando RYR Ca(2+). Le cellule acinose erano appena isolate da rat, pretrattati con etanolo e stimolato con l&#39;agonista muscarinico carbachol (1 &mu;M). Etanolo ha causato un raddoppio nell&#39;attivazione carbachol-indotta della proteasi tripsina e chimotripsina (p &lt; 0,02). Il dantrolene inibitore RYR abolita la valorizzazione delle attivit&agrave; della tripsina e chimotripsina da etanolo (p &lt; 0,005 per entrambe le proteasi). Ulteriormente, etanolo accelerato la velocit&agrave; dell&#39;apicale basolaterale Ca(2+) onda da 9 a 18 &mu;m/s (p &lt; 0,0005; n = cellule/gruppo di 18-22); un aumento nella velocit&agrave; di onda Ca(2+) &egrave; stato osservato anche con un cambiamento da concentrazioni fisiologiche di carbachol (1 &mu;M) ad una concentrazione di supraphysiologic (1 mM) che porta all&#39;attivazione di proteasi. Dantrolene abrogata l&#39;etanolo-indotta accelerazione della velocit&agrave; di onda (p &lt; 0.05; n = 10-16 celle/gruppo). I nostri risultati suggeriscono che il miglioramento dell&#39;attivazione di proteasi patologica di etanolo dipende dai RYR e che un nuovo meccanismo per questo miglioramento pu&ograve; comportare accelerazione RYR-mediata delle onde Ca(2+).

Etanolo potenzia l&#39;attivazione di proteasi carbachol-indotta e accelera Ca2 + onde in acini pancreatici ratto isolato.

I farmaci sono delle principali cause di pancreatite acuta; Tuttavia, poco si sa circa la loro influenza nei bambini. I nostri obiettivi primari sono stati per identificare comuni comorbidit&agrave; ed eziologie concomitante pancreatite nei bambini con pancreatite associati al farmaco. Nostri obiettivi secondari erano per identificare i farmaci pi&ugrave; comunemente associati a diversi gruppi di et&agrave;, valutare le pratiche di gestione e confrontare casi associati al farmaco con casi non associati a farmaci.

Romanzo caratterizzazione di pancreatite associato al farmaco nei bambini.

Medications are a major cause of acute pancreatitis; however, little is known about their influence in children. Our primary aims were to identify common comorbidities and concomitant pancreatitis etiologies in children with drug-associated pancreatitis. Our secondary aims were to identify the most commonly associated drugs in the different age groups, evaluate management practices, and compare drug-associated cases with non-drug-associated cases.

Novel characterization of drug-associated pancreatitis in children.

Les m&eacute;dicaments sont des principales causes de pancr&eacute;atite aigu&euml; ; Cependant, on conna&icirc;t leur influence chez les enfants. Nos principaux objectifs &eacute;taient d&#39;identifier les comorbidit&eacute;s communes et &eacute;tiologies de pancr&eacute;atite concomitante chez les enfants atteints de pancr&eacute;atite associ&eacute;e &agrave; la drogue. Nos objectifs secondaires consistaient &agrave; rechercher les m&eacute;dicaments plus commun&eacute;ment associ&eacute;s &agrave; diff&eacute;rents groupes d&#39;&acirc;ge, &eacute;valuer les pratiques de gestion et comparer les cas li&eacute;s &agrave; un m&eacute;dicament avec cas non associ&eacute;s &agrave; la drogue.

Nouvelle caract&eacute;risation de pancr&eacute;atite associ&eacute;e &agrave; la drogue chez les enfants.

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the Î²-isoform of the catalytic A subunit (CnAÎ²) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAÎ²-deficient mice (CnAÎ²-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAÎ²-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAÎ²-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury.

La pancr&eacute;atite aigu&euml; est un fardeau majeur pour la sant&eacute;, pour lesquels il n&#39;y a actuellement aucune des th&eacute;rapies cibl&eacute;es. Activation pr&eacute;matur&eacute;e des proenzymes digestives ou zymog&egrave;nes, dans les cellules acineuses du pancr&eacute;as est un &eacute;v&eacute;nement pr&eacute;coce et critique dans cette maladie. Une grande amplitude, hausse soutenue des cellules acineuses Ca(2+) est requise pour l&#39;activation de zymog&egrave;ne. Nous avons pr&eacute;c&eacute;demment montr&eacute; dans un mod&egrave;le de pancr&eacute;atite induite par la chol&eacute;cystokinine qu&#39;une cible potentielle de cette signalisation aberrante de Ca(2+) est la calcineurine phosphatase activ&eacute;e par Ca(2+) (Cn). Toutefois, dans cette &eacute;tude, nous avons examin&eacute; le r&ocirc;le du Cn sur l&#39;activation de zymog&egrave;ne tant de blessures, dans la condition clinique pertinente de stimulation neurog&egrave;ne (en donnant le carbachol analogiques de l&#39;ac&eacute;tylcholine) en utilisant trois diff&eacute;rents inhibiteurs de la Cn ou cellules acineuses Cn d&eacute;ficientes. Dans les cellules acineuses souris fra&icirc;chement isol&eacute;es, un pr&eacute;traitement avec FK506, peptide inhibiteur de la calcineurine (CiP) ou cyclosporine (CsA) a bloqu&eacute; activation zymog&egrave;ne intra-acinar (n = 3 ; P &lt; 0,05). Les inhibiteurs de la Cn r&eacute;duit aussi la fuite de la lactate d&eacute;shydrog&eacute;nase (LDH) par 79 %, 62 % et 63 %, respectivement (n = 3 ; P &lt; 0,05). Des diverses isoformes Cn, l&#39;isoforme &beta; de la catalytique une sous-unit&eacute; (CnA&beta;) a &eacute;t&eacute; fortement exprim&eacute;e dans les cellules acineuses de souris. Pour cette raison, nous avons obtenu des cellules acineuses de souris d&eacute;ficientes en CnA&beta; (CnA&beta;-/-) et a observ&eacute; une r&eacute;duction de 84 % et 50 % dans l&#39;activation de la trypsine et la chymotrypsine, respectivement, comparativement aux t&eacute;moins de type sauvage (n = 3 ; P &lt; 0,05). Communiqu&eacute; de la LDH dans les cellules d&eacute;ficientes en CnA&beta; a &eacute;t&eacute; r&eacute;duite de 50 % (n = 2 ; P &lt; 0,05). Les cellules d&eacute;ficientes en CnA&beta; sont &eacute;galement prot&eacute;g&eacute;s contre l&#39;activation de zymog&egrave;ne et la l&eacute;sion cellulaire induite par la c&eacute;rul&eacute;ine analogue de la chol&eacute;cystokinine. Ce qui est important, s&eacute;cr&eacute;tion d&#39;amylase n&#39;est g&eacute;n&eacute;ralement pas affect&eacute;e par les inhibiteurs de la Cn ou Cn carence. Ces donn&eacute;es fournissent des preuves g&eacute;n&eacute;tiques et pharmacologiques qui implique Cn des blessures intra-acinar zymog&egrave;ne activation et cellulaires au cours de la pancr&eacute;atite.

Inhibition pharmacologique et g&eacute;n&eacute;tique de la calcineurine prot&egrave;ge contre l&#39;activation induite par le carbachol de zymog&egrave;ne pathologiques et des l&eacute;sions des cellules acineuses.

Pancreatite acuta &egrave; un onere maggiore salute per cui non ci sono attualmente terapie mirate. Attivazione precoce di proenzymes digestivi, o zimogeni, all&#39;interno delle cellule acinose del pancreas &egrave; un evento precoce e fondamentale in questa malattia. Una grande ampiezza, sostenuto aumento delle cellule acinose Ca(2+) &egrave; richiesto per l&#39;attivazione di zimogeno. Vi abbiamo mostrato in precedenza in un modello di pancreatite indotta colecistochinina che un potenziale bersaglio di questa segnalazione aberrante di Ca(2+) &egrave; il Ca(2+) attivato fosfatasi calcineurina (Cn). Tuttavia, in questo studio, abbiamo esaminato il ruolo di Cn su attivazione zimogeno e del pregiudizio, nella condizione clinicamente rilevante di stimolazione neurogena (fornendo il carbachol analogico acetilcolina) utilizzando tre diversi inibitori Cn o cellule acinose Cn-carenti. Nelle cellule acinose del mouse appena isolato, pretrattamento con FK506, peptidi inibitori della calcineurina (CiP) o ciclosporina (CsA) bloccato attivazione intra-acinar zimogeno (n = 3; P &lt; 0,05). Anche gli inibitori Cn ridotta dispersione della Lattato deidrogenasi (LDH) 79%, 62% e 63%, rispettivamente (n = 3; P &lt; 0,05). Delle diverse isoforme di Cn, l&#39;isoforma &beta; del catalitico A subunit&agrave; (CnA&beta;) era fortemente espresso nelle cellule acinose del mouse. Per questo motivo, abbiamo ottenuto le cellule acinose dal mouse CnA&beta;-carenti (CnA&beta;-/-) e osservate un 84% e 50% riduzione nell&#39;attivazione della tripsina e chimotripsina, rispettivamente, rispetto al wild-type controlli (n = 3; P &lt; 0,05). Rilascio LDH nelle cellule CnA&beta;-carente &egrave; stato ridotto del 50% (n = 2; P &lt; 0,05). Le cellule CnA&beta;-carenti erano protetti anche contro zimogeno attivazione e lesione cellulare indotta dalla colecistochinina analogico caerulein. Soprattutto, la secrezione di amilasi generalmente non fu influenzata dal Cn inibitori o deficit di Cn. Questi dati forniscono prove farmacologiche sia genetica che implica Cn lesioni delle cellule e l&#39;attivazione di zimogeno intra-acinar durante la pancreatite.

Inibizione farmacologica e genetica della calcineurina protegge contro attivazione indotta carbachol patologica zimogeno e lesione delle cellule acinose.

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 Î¼M and a peak-plateau signal at 500 Î¼M, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice.

La pancr&eacute;atite biliaire est l&#39;&eacute;tiologie la plus courante de pancr&eacute;atite aigu&euml;, mais son m&eacute;canisme physiopathologique reste incertaine. Ca(2+) signaux g&eacute;n&eacute;r&eacute;s dans les cellules acineuses du pancr&eacute;as amorcer la phase pr&eacute;coce de la pancr&eacute;atite, et les acides biliaires peuvent d&eacute;clencher la lib&eacute;ration de Ca(2+) intracellulaire anormale de cellules acineuses. Nous avons d&eacute;montr&eacute; pr&eacute;c&eacute;demment que Ca(2+) lib&eacute;r&eacute; via le canal de Ca(2+) intracellulaire, le r&eacute;cepteur &agrave; la ryanodine (RyR), contribue au signal Ca(2+) aberrant. Dans cette &eacute;tude, nous avons examin&eacute; si l&#39;inhibition RyR prot&egrave;ge contre les signaux Ca(2+) pathologique, l&eacute;sions des cellules acineuses et exposition acide biliaire de la pancr&eacute;atite. Le tauro-lithocholique acide de bile acide-3-sulfate (TLC) induit des oscillations intracellulaires de Ca(2+) &agrave; 50 &mu;M et un signal de cr&ecirc;te-plateau &agrave; 500 &mu;M et seulement la blessure de ce dernier induit des cellules acineuses, tel que d&eacute;termin&eacute; par la fuite de la lactate d&eacute;shydrog&eacute;nase (LDH). Un pr&eacute;traitement avec les inhibiteurs de RyR dantrol&egrave;ne ou ryanodine converti le signal de cr&ecirc;te-plateau &agrave; un mod&egrave;le pour la plupart oscillatoire (P 0,05 &lt;). Ils ont aussi r&eacute;duit fuite LDH cellules acineuses, blebbing basolat&eacute;rale et absorption de l&#39;iodure de propidium (P 0,05 &lt;). In vivo, une dose unique de dantrol&egrave;ne (5 mg/kg), 1 h avant, soit 2 h apr&egrave;s la perfusion de TLC intraductal, r&eacute;duit la gravit&eacute; de la pancr&eacute;atite jusqu&#39;au niveau du contr&ocirc;le (P 0,05 &lt;). Ces r&eacute;sultats sugg&egrave;rent que la gravit&eacute; de la pancr&eacute;atite biliaire peut &ecirc;tre am&eacute;lior&eacute;e par l&#39;utilisation clinique des inhibiteurs de RyR.

Les r&eacute;cepteurs de la ryanodine contribuent &agrave; signalisation calcique pathologique induite par l&#39;acide biliaire et pancr&eacute;atite chez les souris.

Pancreatite biliare &egrave; l&#39;eziologia pi&ugrave; comune di pancreatite acuta, ma il suo meccanismo fisiopatologico rimane poco chiaro. CA(2+) segnali generati all&#39;interno delle cellule acinose del pancreas avviare la prima fase della pancreatite, e gli acidi biliari pu&ograve; suscitare rilascio Ca(2+) intracellulare anomala delle cellule acinose. Abbiamo precedentemente dimostrato che Ca(2+) rilasciato tramite il canale Ca(2+) intracellulare, il recettore rianodinico (RyR), contribuisce al segnale Ca(2+) aberrante. In questo studio, abbiamo esaminato se RyR inibizione protegge contro patologica di segnali, lesione delle cellule acinose e pancreatite da acidi biliari esposizione Ca(2+). Gli acidi biliari tauro-lithocholic acido-3-solfato (TLCS) indotta da oscillazioni intracellulari di Ca(2+) a 50 &mu;M e un segnale di picco-altopiano a 500 &mu;M e solo l&#39;infortunio di quest&#39;ultima indotta delle cellule acinose, come determinato dalla perdita di Lattato deidrogenasi (LDH). Pretrattamento con gli inibitori di RyR dantrolene o rianodinico convertito il segnale di picco-altopiano in un modello prevalentemente oscillatorio (P &lt; 0,05). Hanno ridotto anche perdita di cellule acinose LDH, blebbing basolaterale e assorbimento di ioduro di propidium (P &lt; 0,05). In vivo, una singola dose di dantrolene (5 mg/kg), dato 1 h prima o 2 ore dopo infusione TLCS intraductal, ridotto la gravit&agrave; della pancreatite fino al livello del controllo (P &lt; 0,05). Questi risultati suggeriscono che la gravit&agrave; della pancreatite biliare pu&ograve; essere migliorata con l&#39;uso clinico di RyR inibitori.

Rianodinico recettori contribuiscono alla segnalazione di calcio patologica indotta da acidi biliari e pancreatite nei topi.

The association between primary hyperparathyroidism (PHPT) and acute or chronic pancreatitis is controversial. For this reason, we conducted a review of the literature over the past 30 years to explore the relationship between these 2 disorders. Ten retrospective studies each with >50 patients diagnosed with PHPT were identified. With the notable exception of 2 studies, the rate of pancreatitis among patients with PHPT was higher than that reported in general among hospitalized patients without PHPT. A higher serum calcium level may contribute to pancreatitis in these cases, along with additional genetic or environmental insults. Hypercalcemia may predispose the pancreatic acinar cell to abnormal, sustained calcium levels, lead to premature pancreatic protease activation, and pancreatitis. Although there was only short-term follow-up, most reports cited that definitive treatment of PHPT by parathyroidectomy led to the resolution of pancreatitis attacks. The published cohorts of patients with PHPT and pancreatitis are subject to bias, because serum calcium screening was not universally performed among all control nonpancreatitis patients to evaluate for PHPT. However, the pooled clinical and experimental data suggest an association between PHPT and pancreatitis and implicate hypercalcemia. For clinicians, it is important to recognize pancreatitis in patients with PHPT and, conversely, to consider PHPT by checking serum calcium levels in patients, who present with an unexplained pancreatitis.

The association of primary hyperparathyroidism with pancreatitis.

L&#39;association entre l&#39;hyperparathyro&iuml;die primaire (PHPT) et la pancr&eacute;atite aigu&euml; ou chronique est controvers&eacute;e. Pour cette raison, nous avons effectu&eacute; une revue de la litt&eacute;rature sur les 30 derni&egrave;res ann&eacute;es &agrave; explorer la relation entre ces 2 troubles. R&eacute;trospective de dix &eacute;tudes chacun avec &gt; 50 patients diagnostiqu&eacute;s avec PHPT ont &eacute;t&eacute; identifi&eacute;s. &Agrave; l&#39;exception notable des deux &eacute;tudes, le taux de la pancr&eacute;atite chez les patients avec PHPT &eacute;tait sup&eacute;rieur &agrave; celui indiqu&eacute; en g&eacute;n&eacute;ral chez les patients hospitalis&eacute;s sans PHPT. Un taux de calcium s&eacute;rique plus &eacute;lev&eacute; peut-&ecirc;tre contribuer &agrave; la pancr&eacute;atite dans ces cas, ainsi que d&#39;autres insultes g&eacute;n&eacute;tiques ou environnementaux. Hypercalc&eacute;mie peut pr&eacute;disposer les cellules acineuses du pancr&eacute;as &agrave; calc&eacute;mie anormale, soutenue, conduisent &agrave; l&#39;activation de la prot&eacute;ase pancr&eacute;atique pr&eacute;matur&eacute;e et la pancr&eacute;atite. Bien qu&#39;il y avait seulement &agrave; court terme suivi, la plupart des rapports cit&eacute;s que le traitement d&eacute;finitif du PHPT par parathyro&iuml;dectomie a conduit &agrave; la r&eacute;solution des crises de pancr&eacute;atite. Les cohortes publi&eacute;s des patients atteints de pancr&eacute;atite et de PHPT sont impartiales, parce que le d&eacute;pistage de calcium s&eacute;rique n&#39;a pas &eacute;t&eacute; universellement r&eacute;alis&eacute;e chez tous les patients de nonpancreatitis de contr&ocirc;le pour &eacute;valuer pour PHPT. Toutefois, les donn&eacute;es exp&eacute;rimentales et cliniques regroup&eacute;es sugg&egrave;rent une association entre PHPT et pancr&eacute;atite et impliquent une hypercalc&eacute;mie. Pour les cliniciens, il est important de reconna&icirc;tre la pancr&eacute;atite chez les patients avec PHPT et, inversement, d&#39;examiner la PHPT en v&eacute;rifiant le s&eacute;rum calcium niveaux patients, qui pr&eacute;sentent une pancr&eacute;atite inexpliqu&eacute;e.

L&#39;association de l&#39;hyperparathyro&iuml;die primaire ayant une pancr&eacute;atite.

L&#39;associazione tra iperparatiroidismo primario (PHPT) e pancreatite acuta o cronica &egrave; controversa. Per questo motivo, abbiamo condotto una revisione della letteratura degli ultimi anni 30 per esplorare il rapporto tra questi 2 disturbi. Retrospettiva dieci studi ciascuno con &amp;gt; 50 pazienti diagnosticati con PHPT sono stati identificati. Con la notevole eccezione di 2 studi, il tasso di pancreatite tra i pazienti con PHPT era superiore a quello segnalato in generale tra i pazienti ospedalizzati senza PHPT. Un pi&ugrave; alto livello di calcio nel siero possa contribuire alla pancreatite in questi casi, insieme ad ulteriori insulti genetici o ambientali. Ipercalcemia pu&ograve; predisporre il cellule acinose del pancreas a livelli di calcio anormale, sostenuta, portano all&#39;attivazione di proteasi pancreatica prematura e pancreatite. Anche se c&#39;era solo follow-up a breve termine, la maggior parte dei rapporti citati che il trattamento definitivo di PHPT da parathyroidectomy ha portato alla risoluzione degli attacchi di pancreatite. Pubblicato coorti di pazienti con PHPT e pancreatite sono soggetti a bias, perch&eacute; lo screening di calcio siero non era universalmente eseguito tra tutti i pazienti di controllo nonpancreatitis per valutare per PHPT. Tuttavia, i pool dati sperimentali e clinici suggeriscono un&#39;associazione tra PHPT e pancreatite e coinvolgono ipercalcemia. Per i medici, &egrave; importante riconoscere la pancreatite nei pazienti con PHPT e, al contrario, considerare PHPT controllando siero calcio livelli in pazienti che presentano un&#39;inspiegata pancreatite.

L&#39;associazione di iperparatiroidismo primario con pancreatite.

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca(2+). Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-ÎºB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform AÎ² (CnAÎ²) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca(2+) generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin.

La pancr&eacute;atite biliaire est la principale cause de la pancr&eacute;atite aigu&euml; chez les enfants et les adultes. Un m&eacute;canisme propos&eacute; est le reflux de la bile dans le canal pancr&eacute;atique. Acides biliaires exposition cause un pr&eacute;judice de cellules acineuses du pancr&eacute;as par une hausse soutenue des Ca(2+) cytosolique. Ainsi, il serait cliniquement pertinent de savoir les cibles de ce signal aberrant de Ca(2+). Nous avons &eacute;mis l&#39;hypoth&egrave;se que la calcineurine phosphatase activ&eacute;e par Ca(2+) est telle une cible Ca(2+). Pour examiner l&#39;activation de la calcineurine, nous avons infect&eacute; des cellules acineuses primaires de souris avec un ad&eacute;novirus qui exprime le promoteur d&#39;un effecteur de la calcineurine en aval, facteur nucl&eacute;aire des lymphocytes T activ&eacute;s (NFAT). La bile acide taurolithocholique acide-3-sulfate (TLC) a &eacute;t&eacute; principalement utilis&eacute; pour examiner les r&eacute;ponses des acides biliaires. TLCS caus&eacute; activation calcineurine uniquement &agrave; des concentrations qui provoquent des l&eacute;sions des cellules acineuses. L&#39;activation de la calcineurine par TLC a &eacute;t&eacute; abolie par ch&eacute;lation Ca(2+) intracellulaire. Un pr&eacute;traitement avec bis(o-aminophenoxy)-1, 2 &eacute;thane-N, N, N&#39;, N&#39;-t&eacute;traac&eacute;tique (ester ac&eacute;toxym&eacute;thyl) (BAPTA-AM) ou les trois inhibiteurs de la calcineurine sp&eacute;cifiques FK506, cyclosporine A ou peptide inhibiteur de la calcineurine emp&ecirc;ch&eacute; l&eacute;sion induite par l&#39;acide biliaire des cellules acineuses mesur&eacute;e par absorption d&#39;iodure fuite et propidium lactate d&eacute;shydrog&eacute;nase. Les inhibiteurs de la calcineurine r&eacute;duit l&#39;activation intra-acinar du chymotrypsinog&egrave;ne dans les 30 minutes de l&#39;administration de TLC, et ils emp&ecirc;chait &eacute;galement l&#39;activation de NF-&kappa;B. In vivo, les souris qui ont re&ccedil;u le FK506 ou comportaient des lacunes dans la sous-unit&eacute; b&ecirc;ta-amylo&iuml;des (CnA&beta;) de la calcineurine isoforme avaient att&eacute;nu&eacute; la gravit&eacute; de la pancr&eacute;atite apr&egrave;s la perfusion de TLC ou acide taurocholique dans le canal pancr&eacute;atique. En r&eacute;sum&eacute;, nous d&eacute;montrons que des cellules acineuses calcineurine est activ&eacute;e en r&eacute;ponse &agrave; Ca(2+) g&eacute;n&eacute;r&eacute;s par l&#39;exposition de l&#39;acide biliaire, l&eacute;sions pancr&eacute;atiques induites par les acides biliaires d&eacute;pend de l&#39;activation de la calcineurine et inhibiteurs de la calcineurine peuvent pr&eacute;voir un traitement d&#39;appoint pour la pancr&eacute;atite biliaire.

Les acides biliaires induisent des l&eacute;sions des cellules acineuses du pancr&eacute;as et la pancr&eacute;atite en activant la calcineurine.

Pancreatite biliare &egrave; la principale causa di pancreatite acuta in adulti e bambini. Un meccanismo proposto &egrave; il reflusso di bile nel dotto pancreatico. Acido di bile esposizione provoca lesione delle cellule acinose del pancreas attraverso una crescita sostenuta in Ca(2+) citosolico. Quindi, sarebbe clinicamente rilevante conoscere gli obiettivi di questo aberrante segnale Ca(2+). Abbiamo ipotizzato che il Ca(2+) attivato fosfatasi calcineurina &egrave; una destinazione di Ca(2+). Per esaminare l&#39;attivazione della calcineurina, siamo infetti cellule acinose primarie da topi con un adenovirus esprimendo il promotore per un effector downstream calcineurina, fattore nucleare delle cellule T attivate (NFAT). L&#39;acido di bile taurolithocholic acido-3-solfato (TLCS) &egrave; stato utilizzato principalmente per esaminare le risposte degli acidi biliari. TLCS causato calcineurina attivazione solo a concentrazioni che causano lesioni delle cellule acinose. L&#39;attivazione della calcineurina da TLCS fu abolito da chelanti Ca(2+) intracellulare. Pretrattamento con 1,2-bis(o-aminophenoxy) etano-N, N, N&#39;, N&#39;-l&#39;acido etilendiamminotetraacetico (estere acetossimetil) (BAPTA-AM) o i tre inibitori della calcineurina specifico FK506, ciclosporina A o peptidi inibitori della calcineurina evitato lesioni biliari acido-indotta delle cellule acinose come misurato da assorbimento di ioduro di perdite e propidium Lattato deidrogenasi. Gli inibitori della calcineurina riduce l&#39;attivazione intra-acinar di chimotripsinogeno entro 30 min di amministrazione TLCS, e hanno impedito anche attivazione di NF-&kappa;B. In vivo, topi che ricevuto FK506 o erano carenti nella subunit&agrave; della calcineurina isoforma A&beta; (CnA&beta;) erano ridotta gravit&agrave; pancreatite dopo infusione di Acido taurocolico o TLCS nel dotto pancreatico. In sintesi, ci dimostrano che cellule acinose calcineurina &egrave; attivata in risposta a Ca(2+) generati dall&#39;esposizione degli acidi biliari, lesioni del pancreas indotta da acido di bile dipende dall&#39;attivazione della calcineurina e inibitori della calcineurina pu&ograve; fornire una terapia aggiuntiva per la pancreatite biliare.

Acidi biliari indurre lesioni delle cellule acinose del pancreas e pancreatite attivando calcineurina.

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca(2+). These aberrant Ca(2+) elevations are triggered by release of Ca(2+) from apical Ca(2+) pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca(2+) release channel (IP3R2(-/-)). Using live acinar cell Ca(2+) imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca(2+) signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2(-/-) and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca(2+) signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

IP3 receptor type 2 deficiency is associated with a secretory defect in the pancreatic acinar cell and an accumulation of zymogen granules.

La pancr&eacute;atite aigu&euml; est une maladie mortelle douloureuse du pancr&eacute;as dont l&#39;&eacute;tiologie est souvent multifactorielle. Il est d&#39;une grande importance pour comprendre l&#39;interaction entre les facteurs qui pr&eacute;disposent les patients afin de d&eacute;velopper la maladie. Un de ces facteurs est une &eacute;l&eacute;vation excessive des cellules acineuses du pancr&eacute;as Ca(2+). Ces &eacute;l&eacute;vations Ca(2+) aberrantes sont d&eacute;clench&eacute;es par la lib&eacute;ration de Ca(2+) partir d&#39;un m&eacute;lange de Ca(2+) apical qui sont bloqu&eacute;es par les types de r&eacute;cepteurs (IP3R) inositol 1,4,5-trisphosphate 2 et 3. Dans cette &eacute;tude, nous avons examin&eacute; le r&ocirc;le de IP3R de type 2 (IP3R2) &agrave; l&#39;aide de souris d&eacute;ficientes en ce canal de diffusion de le Ca(2+) (IP3R2(-/-)). Imagerie Ca(2+) des cellules acineuses vivantes, nous avons constat&eacute; que la perte de IP3R2 r&eacute;duit l&#39;amplitude du signal Ca(2+) apical et a retard&eacute; son initiation. Cela a &eacute;t&eacute; associ&eacute;e &agrave; une r&eacute;duction de la lib&eacute;ration d&#39;amylase stimul&eacute;e par le carbachol et une accumulation de zymog&egrave;ne (ZGs). Plus pr&eacute;cis&eacute;ment, il y avait une augmentation du nombre de ZGs 2 fois (P &lt; 0,05) et une expansion de l&#39;aire de piscine ZG au sein de la cellule. Il y a aussi une augmentation de 1.6 et 2.6 fois en amylase cellulaire et le trypsinog&egrave;ne, respectivement. Cependant, les souris n&#39;avaient pas de preuve de dommage du pancr&eacute;as au d&eacute;part, autre qu&#39;un niveau d&#39;amylase s&eacute;rique. De plus, r&eacute;sultats de pancr&eacute;atite en utilisant un mod&egrave;le d&#39;hyperstimulation c&eacute;rul&eacute;ine doux &eacute;taient similaires entre les IP3R2(-/-) et les souris de type sauvage. En r&eacute;sum&eacute;, IP3R2 module signaux Ca(2+) cellules acineuses apicale et la s&eacute;cr&eacute;tion des enzymes pancr&eacute;atiques. Cellules acineuses IP3R d&eacute;ficientes accumulent ZGs, mais les souris ne succombent pas aux dommages pancr&eacute;atiques ou pire des r&eacute;sultats de pancr&eacute;atite.

D&eacute;ficit de type 2 r&eacute;cepteurs IP3 est associ&eacute; &agrave; un d&eacute;faut de s&eacute;cr&eacute;tion dans les cellules acineuses du pancr&eacute;as et une accumulation de zymog&egrave;ne.

Pancreatite acuta &egrave; un disturbo doloroso e life-threatening del pancreas cui eziologia &egrave; spesso multifattoriale. &Egrave; di grande importanza per comprendere l&#39;interazione tra i fattori che predispongono i pazienti a sviluppare la malattia. Un tale fattore &egrave; un aumento eccessivo delle cellule acinose del pancreas Ca(2+). Queste elevazioni Ca(2+) aberranti sono attivate dal rilascio di Ca(2+) dalle piscine Ca(2+) apicale che sono dipendenti dai tipi di recettori (IP3R) inositolo 1.4.5-trifosfato 2 e 3. In questo studio, abbiamo esaminato il ruolo di IP3R tipo 2 (IP3R2) usando i mouse carenti in questo canale di rilascio del Ca(2+) (IP3R2(-/-)). Utilizzando imaging Ca(2+) diretta delle cellule acinose abbiamo scoperto che la perdita di IP3R2 ridotto l&#39;ampiezza del segnale Ca(2+) apicale e ha causato un ritardo nella sua iniziazione. Questa &egrave; stata associata con una riduzione del rilascio di amilasi carbachol-stimolato e un accumulo di granuli di zimogeno (ZGs). In particolare, c&#39;&egrave; stato un aumento di 2 volte il numero di ZGs (P &lt; 0,05) e un&#39;espansione della zona piscina ZG all&#39;interno della cellula. C&#39;era anche un 1.6 - 2.6 volte aumento amilasi cellulare e tripsinogeno, rispettivamente. Tuttavia, i topi non hanno evidenza di lesioni del pancreas al basale, diverso da un livello di amilasi sierica elevata. Ulteriormente, esiti pancreatite utilizzando un modello di iperstimolazione lieve caerulein erano simili tra IP3R2(-/-) e topi wild type. In sintesi, IP3R2 modula segnali Ca(2+) apicale delle cellule acinose e la secrezione di enzimi pancreatici. IP3R-deficienti cellule acinose accumulano ZGs, ma i topi non soccombere ai danni del pancreas o peggio esiti pancreatite.

Deficit di tipo 2 recettore IP3 &egrave; associato un difetto secretivo nel cellule acinose del pancreas e un accumulo di granuli di zimogeno.

Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30-60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca2+ signals in acinar cells as well as nuclear translocation of NF-ÎºB. In this study, we examined the role of the downstream Ca2+ target calcineurin on NF-ÎºB translocation. Freshly isolated mouse acinar cells were infected for 24 hr with an adenovirus expressing an NF-ÎºB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate (TLCS) caused NF-ÎºB activation at concentrations (500 uM) that were associated with cell injury. We show that the NF-ÎºB inhibitor Bay 11-7082 (1 uM) blocked translocation and injury. Pretreatment with the Ca2+ chelator BAPTA, the calcineurin inhibitors, FK506 and cyclosporine A, or use of acinar cells from calcineurin AÎ²-deficient mice each led to reduced NF-ÎºB activation with TLCS. Importantly, these maniuplations did not affect LPS-induced NF-ÎºB activation. A critical upstream regulator of NF-ÎºB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-Î´ isoform. In summary, bile-induced NF-ÎºB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.

Pancreatic acinar cell NF-ÎºB activation due to bile acid exposure is dependent on calcineurin.

La pancr&eacute;atite biliaire est l&#39;&eacute;tiologie la plus courante de pancr&eacute;atite aigu&euml;, avec 30-60 % des cas. Une th&eacute;orie dominante pour le d&eacute;veloppement de la pancr&eacute;atite biliaire est le reflux de la bile dans le canal pancr&eacute;atique et l&#39;exposition subs&eacute;quente &agrave; cellules acineuses du pancr&eacute;as. Les acides biliaires sont connus pour induire des signaux de Ca2 + aberrants dans les cellules acineuses ainsi que la translocation nucl&eacute;aire du NF-&kappa;B. Dans cette &eacute;tude, nous avons examin&eacute; le r&ocirc;le de la cible calcineurine de Ca2 + en aval sur la translocation de NF-&kappa;B. Les cellules acineuses souris fra&icirc;chement isol&eacute;es ont &eacute;t&eacute; infect&eacute;s pendant 24 heures avec un ad&eacute;novirus qui exprime un rapporteur lucif&eacute;rase de NF-&kappa;B. La bile acide taurolithocholique acide-3-sulfate (TLC) caus&eacute; d&#39;activation de NF-&kappa;B aux concentrations (500 uM) qui &eacute;taient associ&eacute;s &agrave; des l&eacute;sions des cellules. Nous montrons que l&#39;inhibiteur de NF-&kappa;B Bay 11-7082 (1 uM) bloqu&eacute; translocation et blessures. Un pr&eacute;traitement avec le ch&eacute;lateur du Ca2 + BAPTA, les inhibiteurs de la calcineurine, FK506 et cyclosporine A ou utilisation des cellules acineuses de la calcineurine souris A&beta;-d&eacute;ficientes chaque conduit &agrave; l&#39;activation de NF-&kappa;B r&eacute;duite avec TLC. Ce qui est important, ces maniuplations n&#39;affecte pas d&#39;activation de NF-&kappa;B induite par LPS. Un critique r&eacute;gulateur en amont de l&#39;activation de NF-&kappa;B est la prot&eacute;ine kinase C, lequel translocation aux membranes des organites diff&eacute;rents dans l&#39;&eacute;tat actif. Nous d&eacute;montrons que l&#39;inhibition pharmacologique et g&eacute;n&eacute;tique de la calcineurine translocation de blocs de l&#39;isoforme de la PKC-&delta;. En r&eacute;sum&eacute;, bile induite par l&#39;activation de NF-&kappa;B et cellules acineuses blessures sont v&eacute;hicul&eacute;s par la calcineurine, et un m&eacute;canisme pour cette r&eacute;ponse inflammatoire d&eacute;but important semble &ecirc;tre en amont au niveau de la translocation de la PKC.

Les cellules acineuses du pancr&eacute;as activation de NF-&kappa;B dus &agrave; l&#39;exposition des acides biliaires sont tributaire de la calcineurine.

Article	Year
Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 20448143	2010
A comparison of presentation and management trends in acute pancreatitis between infants/toddlers and older children. Journal of pediatric gastroenterology and nutrition\| PubMed ID: 20479687	2010
Protease activation during in vivo pancreatitis is dependent on calcineurin activation. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 20501444	2009
Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini. The Journal of biological chemistry\| PubMed ID: 21372126	2011
Novel characterization of drug-associated pancreatitis in children. Journal of pediatric gastroenterology and nutrition\| PubMed ID: 21681111	2011
Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 22323127	2012
Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 22517774	2012
The association of primary hyperparathyroidism with pancreatitis. Journal of clinical gastroenterology\| PubMed ID: 22874807	2012
Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin. The Journal of biological chemistry\| PubMed ID: 23148215	2013
IP3 receptor type 2 deficiency is associated with a secretory defect in the pancreatic acinar cell and an accumulation of zymogen granules. PloS one\| PubMed ID: 23185258	2012
Pancreatic acinar cell NF-ÎºB activation due to bile acid exposure is dependent on calcineurin. The Journal of biological chemistry\| PubMed ID: 23744075	2013

Abrahim I. Orabi

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