IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP3 receptors /IP3R) via its coiled-coil domain and modulates Ca2+ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca2+ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP3 receptors and its impact on intracellular Ca2+ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP3R and altered Ca2+ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca2+, suggesting that IRAG2 leads to activation of IP3R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca2+ signaling, which regulates exocrine pancreatic function.

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Agonist-dependent Phosphorylation of the Inositol 1,4,5-Trisphosphate Receptor

The Journal of General Physiology ◽

10.1085/jgp.113.6.851 ◽

1999 ◽

Vol 113 (6) ◽

pp. 851-872 ◽

Cited By ~ 75

Author(s):

Andrew P. LeBeau ◽

David I. Yule ◽

Guy E. Groblewski ◽

James Sneyd

Keyword(s):

Acinar Cells ◽

Calcium Oscillations ◽

Pancreatic Acinar Cells ◽

Ip3 Receptors ◽

Ip3 Receptor ◽

Open Probability ◽

Probability Curve ◽

Long Period ◽

Inositol 1,4,5 Trisphosphate ◽

S Period

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10–12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675–2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81–84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.

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Inhibitory effect of high leucine concentration on α-amylase secretion by pancreatic acinar cells: possible key factor of proteasome

Bioscience Reports ◽

10.1042/bsr20181455 ◽

2018 ◽

Vol 38 (6) ◽

Cited By ~ 2

Author(s):

Long Guo ◽

Baolong Liu ◽

Chen Zheng ◽

Hanxun Bai ◽

Hao Ren ◽

...

Keyword(s):

Amylase Activity ◽

Acinar Cells ◽

Inhibitory Effect ◽

Proteasome Activity ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

High Concentration ◽

Amylase Release ◽

Cholecystokinin Receptor ◽

Key Factor

The present study aimed to investigate whether leucine affects the pancreatic exocrine by controlling the antisecretory factor (AF) and cholecystokinin receptor (CCKR) expression as well as the proteasome activity in pancreatic acinar cells of dairy calves. The pancreatic acinar cells were isolated from newborn Holstein bull calves and cultured using the Dulbecco’s modified Eagle’s medium/nutrient mixture F12 Ham’s liquid (DMEM/F12). There were six treatments of leucine dosage including 0 (control), 0.23, 0.45, 1.35, 4.05, and 12.15 mM, respectively. After culture for 3 h, the samples were collected for subsequent analysis. As the leucine concentration increased from 0 to 1.35 mM, the α-amylase activity in media decreased significantly (P<0.05), while further increase in leucine concentration did not show any decrease in α-amylase activity. Addition of leucine inhibited (P<0.05) the expression of AF and CCKR, and decreased the activity of proteasome (P<0.05) by 76%, 63%, 24%, 7%, and 9%, respectively. Correlation analysis results showed α-amylase secretion was negatively correlated with leucine concentration (P<0.01), and positively correlated with proteasome activity (P<0.01) and the expression of CCK1R (P<0.01) and AF (P<0.05). The biggest regression coefficient was showed between α-amylase activity and proteasome (0.7699, P<0.001). After inhibition of proteasome by MG-132, low dosage leucine decreased (P<0.05) the activity of proteasome and α-amylase, as well as the expression of CCK1R. In conclusion, we demonstrated that the high-concentration leucine induced decrease in α-amylase release was mainly by decreasing proteasome activity.

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Regulation of CCK-induced amylase release by PKC-δ in rat pancreatic acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00111.2004 ◽

2004 ◽

Vol 287 (4) ◽

pp. G764-G771 ◽

Cited By ~ 40

Author(s):

Chenwei Li ◽

Xuequn Chen ◽

John A. Williams

Keyword(s):

Acinar Cells ◽

Adenoviral Vectors ◽

Dominant Negative ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Wild Type ◽

Amylase Release ◽

Pkc Isoforms ◽

Chemical Inhibitors ◽

Dominant Negative Variant

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, α, δ, ε, and ζ, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-α, -δ, and -ε, but not -ζ from soluble to membrane fraction. CCK-induced amylase release was inhibited ∼30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-δ inhibitor, but not by Gö6976, a PKC-α inhibitor, at concentrations from 1 to 5 μM. Neither overexpression of wild-type or dominant-negative PKC-α affected CCK-induced amylase release. Overexpression of PKC-δ and -ε enhanced amylase release, whereas only dominant-negative PKC-δ inhibited amylase release by 25%. PKC-δ overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-δ only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-δ and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-δ but had no further effect in the presence of dominant-negative PKC-δ. These results indicate that PKC-δ is the PKC isoform involved with amylase secretion.

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Morphometric Measurements to Quantify the Cerulein Induced Hyperstimulatory Pancreatitis of Rats under the Protective Effect of Lectins

Analytical Cellular Pathology ◽

10.1155/1998/618913 ◽

1998 ◽

Vol 17 (4) ◽

pp. 219-230 ◽

Cited By ~ 6

Author(s):

Ludwig Jonas ◽

Ulrike Mikkat ◽

Anke Witte ◽

Uta Beckmann ◽

Katrin Dölker ◽

...

Keyword(s):

Protective Effect ◽

Amylase Activity ◽

Acinar Cells ◽

Inhibitory Effect ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Ulex Europaeus ◽

Serum Amylase Activity

In preceding papers we demonstrated an inhibitory effect of wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA) on the cholecystokinin (CCK) binding to the CCK receptor of rat pancreatic cells and also on the CCK induced Ca2+release and α-amylase secretionin vitroas well as on pancreatic secretion of intact ratsin vivo. In the present study we show the same inhibitory effect of both lectins on the cerulein pancreatitis of rats. This acute pancreatitis was induced by supramaximal injections (5 µg/kg/h iv or 10 µg/kg/h ip) of the CCK analogue cerulein in rats every hour. To monitor the degree of pancreatitis, we measured the number and diameter of injury vacuoles in the pancreatic acinar cells as one of the most important signs of this type of pancreatitis by light microscopic morphometry with two different systems on paraffin sections. Furthermore, the serum α-amylase activity was measured biochemically. We found a correlation between the diameter of vacuoles inside the acinar cells and the serum enzyme activity up to 24 h. The simultaneous ip administration of cerulein and WGA or UEA in a dosage of 125 µg/kg/h for 8 h led to a reduction of vacuolar diameter from 13.1 ± 2.0 µm (cerulein) to 7.5 ± 1.1 µm (cerulein + WGA) or 7.2 ± 1.3 µm (cerulein + UEA). The serum amylase activity was reduced from 63.7 ± 15.8 mmol/l \times min (cerulein) to 37.7 ± 11.8 (cerulein + WGA) or 39.4; +52.9; -31.1 (cerulein + UEA-I). Both parameters allow the grading this special type of pancreatitis to demonstrate the protective effect of the lectins.

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Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+entry in mouse pancreatic acinar cells

AJP Cell Physiology ◽

10.1152/ajpcell.00241.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. C214-C221 ◽

Cited By ~ 19

Author(s):

Juan A. Rosado ◽

Pedro C. Redondo ◽

Ginés M. Salido ◽

Stewart O. Sage ◽

Jose A. Pariente

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Secretory Cell ◽

Botulinum Toxin A ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Secretory Proteins ◽

Amylase Secretion ◽

Cell Type ◽

Store Depletion

We recently reported that store-operated Ca2+entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

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Regulation by diacylglycerol of calcium-evoked amylase secretion from intact and permeabilized pancreatic acinar cells

Cell Calcium ◽

10.1016/0143-4160(90)90025-p ◽

1990 ◽

Vol 11 (8) ◽

pp. 501-506 ◽

Cited By ~ 3

Author(s):

T. Komabayashi ◽

J.S. McKinney ◽

R.P. Rubin

Keyword(s):

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Amylase Secretion

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Enhanced Secretion of Amylase from Exocrine Pancreas of Connexin32-deficient Mice

The Journal of Cell Biology ◽

10.1083/jcb.141.5.1267 ◽

1998 ◽

Vol 141 (5) ◽

pp. 1267-1275 ◽

Cited By ~ 52

Author(s):

Marc Chanson ◽

Marjorie Fanjul ◽

Domenico Bosco ◽

Eric Nelles ◽

Susanne Suter ◽

...

Keyword(s):

Acinar Cell ◽

Intercellular Communication ◽

Plaque Assay ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Wild Type ◽

Patch Clamp Recording ◽

Junctional Communication

To determine whether junctional communication between pancreatic acinar cells contributes to their secretory function in vivo, we have compared wild-type mice, which express the gap junctional proteins connexin32 (Cx32) and connexin26, to mice deficient for the Cx32 gene. Pancreatic acinar cells from Cx32 (−/−) mice failed to express Cx32 as evidenced by reverse transcription–PCR and immunolabeling and showed a marked reduction (4.8- and 25-fold, respectively) in the number and size of gap junctions. Dye transfer studies showed that the extent of intercellular communication was inhibited in Cx32 (−/−) acini. However, electrical coupling was detected by dual patch clamp recording in Cx32 (−/−) acinar cell pairs. Although wild-type and Cx32 (−/−) acini were similarly stimulated to release amylase by carbamylcholine, Cx32 (−/−) acini showed a twofold increase of their basal secretion. This effect was caused by an increase in the proportion of secreting acini, as detected with a reverse hemolytic plaque assay. Blood measurements further revealed that Cx32 (−/−) mice had elevated basal levels of circulating amylase. The results, which demonstrate an inverse relationship between the extent of acinar cell coupling and basal amylase secretion in vivo, support the view that the physiological recruitment of secretory acinar cells is regulated by gap junction mediated intercellular communication.

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Cholecystokinin octapeptide inhibits Ca2+-dependent amylase secretion from permeabilized pancreatic acini by blocking the MgATP-dependent priming of exocytosis

Biochemical Journal ◽

10.1042/bj3300329 ◽

1998 ◽

Vol 330 (1) ◽

pp. 329-334 ◽

Cited By ~ 8

Author(s):

J. Philip PADFIELD ◽

Ninder PANESAR

Keyword(s):

Acinar Cells ◽

Secretory Response ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Rapid Phase ◽

Pancreatic Acini ◽

Cholecystokinin Octapeptide ◽

Cholecystokinin Receptor ◽

Specific Antagonist

At present little is known about how the low-affinity cholecystokinin receptor inhibits secretagogue-stimulated amylase secretion from pancreatic acinar cells. To examine this question we have determined how cholecystokinin octapeptide (CCK8) influences Ca2+-dependent amylase secretion from α-toxin-permeabilized pancreatic acini. CCK8 significantly inhibited Ca2+-stimulated amylase secretion. The inhibitory actions of CCK8 were completely blocked by the addition of JMV-180, a specific antagonist for the low-affinity CCK8 receptor. Previous studies have shown that Ca2+-dependent amylase secretion from α-toxin-permeabilized acini has two distinct phases [Padfield and Panesar (1997) Am. J. Physiol. 36, G655-660]. There is an initial rapid phase of secretion which represents release from exocytotic sites primed by MgATP prior to permeabilization. This is followed by a slower sustained phase of secretion which, in part, reflects the MgATP-dependent repriming of the exocytotic machinery. CCK8 did not influence the initial rapid phase of the Ca2+-dependent secretory response, but inhibited the second slower sustained phase. Moreover, CCK8 was shown to inhibit the MgATP-dependent priming of exocytosis in the acini. These results indicate that the low-affinity CCK receptor blocks stimulated amylase secretion by inhibiting the MgATP-dependent repriming of exocytosis.

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