InsP3 receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2+ entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP3Rs (InsP3 receptors). The co-localization between Orai1 and IP3Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2+ store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2+ store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP3Rs and a basolateral pool that interacts with STIM1 following the Ca2+ store depletion. Experiments on IP3R knockout animals demonstrated that the apical Orai1 localization does not require IP3Rs and that IP3Rs are not necessary for the activation of SOCE. However, the InsP3-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP3Rs could have an inhibitory effect on this Ca2+ entry mechanism

An interferon-like small chemical compound CDM-3008 suppresses hepatitis B virus through induction of interferon-stimulated genes

B型肝炎ウイルス抑制物質の作用機序解明 --新規抗B型肝炎治療薬の開発へ期待--. 京都大学プレスリリース. 2019-06-13.Oral administration of nucleotide analogues and injection of interferon-α (IFNα) are used to achieve immediate suppression in replication of hepatitis B virus (HBV). Nucleotide analogs and IFNα inhibit viral polymerase activity and cause long-term eradication of the virus at least in part through removing covalently closed circular DNA (cccDNA) via induction of the APOBEC3 deaminases family of molecules, respectively. This study aimed to explore whether the orally administrable low molecular weight agent CDM-3008 (RO8191), which mimics IFNα through the binding to IFNα/β receptor 2 (IFNAR2) and the activation of the JAK/STAT pathway, can suppress HBV replication and reduce cccDNA levels. In primary cultured human hepatocytes, HBV DNA levels were decreased after CDM-3008-treatment in a dose-dependent manner with a half-maximal inhibitory concentration (IC50) value of 0.1 μM, and this was accompanied by significant reductions in cellular cccDNA levels, both HBeAg and HBsAg levels in the cell culture medium. Using a microarray we comprehensively analyzed and compared changes in gene (mRNA) expression in CDM-3008- and IFNα-treated primary cultured human hepatocytes. As reported previously, CDM-3008 mimicked the induction of genes that participate in the interferon signaling pathway. OAS1 and ISG20 mRNA expression was similarly enhanced by both CDM-3008 and IFNα. Thus, CDM-3008 could suppress pgRNA expression to show anti-HBV activity. APOBEC3F and 3G mRNA expression was also induced by CDM-3008 and IFNα treatments, suggesting that cccDNA could be degraded through induced APOBEC3 family proteins. We identified the genes whose expression was specifically enhanced in CDM-3008-treated cells compared to IFNα-treated cells. The expression of SOCS1, SOCS2, SOCS3, and CISH, which inhibit STAT activation, was enhanced in CDM-3008-treated cells suggesting that a feedback inhibition of the JAK/STAT pathway was enhanced in CDM-3008-treated cells compared to IFNα-treated cells. In addition, CDM-3008 showed an additive effect with a clinically-used nucleoside entecavir on inhibition of HBV replication. In summary, CDM-3008 showed anti-HBV activity through activation of the JAK/STAT pathway, inducing the expression of interferon-stimulated genes (ISGs), with greater feedback inhibition than IFNα

Mice lacking inositol 1,4,5-trisphosphate receptors exhibit dry eye.

Tear secretion is important as it supplies water to the ocular surface and keeps eyes moist. Both the parasympathetic and sympathetic pathways contribute to tear secretion. Although intracellular Ca2+ elevation in the acinar cells of lacrimal glands is a crucial event for tear secretion in both the pathways, the Ca2+ channel, which is responsible for the Ca2+ elevation in the sympathetic pathway, has not been sufficiently analyzed. In this study, we examined tear secretion in mice lacking the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3 (Itpr2-/-;Itpr3-/-double-knockout mice). We found that tear secretion in both the parasympathetic and sympathetic pathways was abolished in Itpr2-/-;Itpr3-/- mice. Intracellular Ca2+ elevation in lacrimal acinar cells after acetylcholine and epinephrine stimulation was abolished in Itpr2-/-;Itpr3-/- mice. Consequently, Itpr2-/-;Itpr3-/- mice exhibited keratoconjunctival alteration and corneal epithelial barrier disruption. Inflammatory cell infiltration into the lacrimal glands and elevation of serum autoantibodies, a representative marker for Sjögren's syndrome (SS) in humans, were also detected in older Itpr2-/-;Itpr3-/- mice. These results suggested that IP3Rs are essential for tear secretion in both parasympathetic and sympathetic pathways and that Itpr2-/-;Itpr3-/- mice could be a new dry eye mouse model with symptoms that mimic those of SS

Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors

Toxic alcohol effects on pancreatic acinar cells, causing the often fatal human disease acute pancreatitis, are principally mediated by fatty acid ethyl esters (non-oxidative products of alcohol and fatty acids), emptying internal stores of Ca2+. This excessive Ca2+ liberation induces Ca2+-dependent necrosis due to intracellular trypsin activation. Our aim was to identify the specific source of the Ca2+ release linked to the fatal intracellular protease activation. In 2-photon permeabilized mouse pancreatic acinar cells, we monitored changes in the Ca2+ concentration in the thapsigargin-sensitive endoplasmic reticulum (ER) as well as in a bafilomycin-sensitive acid compartment, localized exclusively in the apical granular pole. We also assessed trypsin activity in the apical granular region. Palmitoleic acid ethyl ester (POAEE) elicited Ca2+ release from both the ER as well as the acid pool, but trypsin activation depended predominantly on Ca2+ release from the acid pool, that was mainly mediated by functional inositol 1,4,5- trisphosphate receptors (IP3Rs) of types 2 and 3. POAEE evoked very little Ca2+ release and trypsin activation when IP3Rs of both types 2 and 3 were knocked out. Antibodies against IP3Rs of types 2 and 3, but not type 1, markedly inhibited POAEE-elicited Ca2+ release and trypsin activation. We conclude that Ca2+ release through IP3Rs of types 2 and 3 in the acid granular Ca2+ store induces intracellular protease activation, and propose that this is a critical process in the initiation of alcohol-related acute pancreatitis

Histological analysis of lacrimal gland tissues.

<p>(A) Tissue sections of lacrimal glands from wild-type and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> mice were stained by hematoxylin/eosin (HE) and observed under light microscopy. White arrowheads indicate inflammatory infiltrates. Scale bar: 50 µm. (B) Electron micrographs of lacrimal glands from wild-type and <i>Itpr2^−/−</i>; <i>Itpr3^−/−</i> mice. Scale bar: upper panels, 5 µm; lower panels, 2 µm. All experiments were performed at least three times, and representative data are shown. N; Nucleus, lu; lumen, ER; endoplasmic reticulum. (C) Relative lacrimal acinar cell area. The acinar cell area of wild-type (n = 54) and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> (n = 59) lacrimal acinar cells was measured using HE-stained sections. Values represent the means ± SEM. Student’s t-test, ***, P<0.001.</p

Defects in tear secretion in <i>Itpr2^−/−;Itpr3^−/−</i> mice via both parasympathetic and sympathetic pathways.

<p>(A) Tear volume in wild-type (n = 12) and <i>Itpr^−/−</i> (n = 16) mice within 15 min of pilocarpine stimulation. (B) Average body weight of wild-type and the <i>Itpr^−/−</i> mice at 6 weeks. (C) Average lacrimal gland weights of wild-type and the <i>Itpr^−/−</i> mice. (D) Tear secretion by pilocarpine adjusted for the weight of each lacrimal gland. (E) Time course of tear secretion in each 5-min period after pilocarpine administration in wild-type (diamond), <i>Itpr2^−/−</i> (triangle), <i>Itpr3^−/−</i> (cross), and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> (square) mice. (F) The tear secretion by epinephrine adjusted for weight of the each lacrimal gland. All data are presented as means ± standard error of the mean (SEM). Student’s t-test, *P<0.05; **P<0.01. All experiments were performed at least three times, and representative data are shown.</p

Altered ocular surface in <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> mice.

<p>(A) Anterior segment photos of the ocular surface. Wild-type and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> mice corneas were viewed and photographed under white light. Debris is indicated by white arrowheads. Bar: 1 mm. (B) Histological detection of conjunctiva mucins stained with periodic acid-Schiff base. The conjunctiva of <i>Itpr2^−/−</i>; <i>Itpr3^−/−</i> mice had abundant mucin complexes (arrow head). Scale bar: 50 µm. (C, D) Anterior segment photos of ocular surface fluorescein staining, and the score. Bar: 1 mm. (E) Comparison of spontaneous blink rate. All data are presented as means ± SEM. Student’s t-test, *P<0.05. All experiments were performed at least three times, and representative data are shown.</p

Infiltration of inflammatory mononuclear cells in <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> lacrimal glands.

<p>(A) Immunostaining of CD45, F4/80, CD19, CD8 and CD4 in lacrimal gland tissue sections from wild-type and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i> mice. White arrowheads indicate inflammatory mononuclear cells. (B) Quantification of TNF-α mRNA expression levels by real time RT-PCR. Six week-old mice; wild-type: n = 8 and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i>: n = 8. Ten week-old mice; wild-type: n = 16, <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i>: n = 10. Mann–Whitney <i>U</i>-test, ***P<0.001, *P<0.05. All data are presented as means ± SEM. (C) Quantification of IL-6 mRNA expression levels by real time RT-PCR. Six week-old mice; wild-type: n = 8 and <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i>: n = 8. Ten week-old mice; wild-type: n = 16, <i>Itpr2^−/−</i>;<i>Itpr3^−/−</i>: n = 10. Mann–Whitney <i>U</i>-test, ***P<0.001. All data are presented as means ± SEM.</p