Aberrant calcium signaling by transglutaminase-mediated posttranslational modification of inositol 1,4,5-trisphosphate receptors.

The inositol 1,4,5-trisphosphate receptor (IP3R) in the endoplasmic reticulum mediates calcium signaling that impinges on intracellular processes. IP3Rs are allosteric proteins comprising four subunits that form an ion channel activated by binding of IP3 at a distance. Defective allostery in IP3R is considered crucial to cellular dysfunction, but the specific mechanism remains unknown. Here we demonstrate that a pleiotropic enzyme transglutaminase type 2 targets the allosteric coupling domain of IP3R type 1 (IP3R1) and negatively regulates IP3R1-mediated calcium signaling and autophagy by locking the subunit configurations. The control point of this regulation is the covalent posttranslational modification of the Gln2746 residue that transglutaminase type 2 tethers to the adjacent subunit. Modification of Gln2746 and IP3R1 function was observed in Huntington disease models, suggesting a pathological role of this modification in the neurodegenerative disease. Our study reveals that cellular signaling is regulated by a new mode of posttranslational modification that chronically and enzymatically blocks allosteric changes in the ligand-gated channels that relate to disease states.

LC/ESI-MS/MS method for determination of salivary eicosapentaenoic acid concentration to arachidonic acid concentration ratio.

A simple liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for determination of the eicosapentaenoic acid (EPA) concentration to arachidonic acid (AA) concentration ratio in human saliva has been developed. The EPA/AA ratio in serum or plasma is widely recognized as a useful indicator in identifying the risk of cardiovascular disease, especially atherosclerosis. The salivary EPA/AA ratio is expected to be a convenient alternative to the serum or plasma EPA/AA ratio, because saliva offers the advantages of easy and noninvasive sampling. The saliva was deproteinized with acetonitrile, purified using an Oasis HLB cartridge, and derivatized with 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ). The derivatized EPA and AA were subjected to LC/ESI-MS/MS, and the EPA/AA ratio was determined using the selected reaction monitoring mode. The DAPPZ-derivatization increased the ESI sensitivity by 100- and 300-fold for EPA and AA, respectively, and enabled the detection of trace fatty acids in saliva using a 200 µL sample. The assay reproducibility was satisfactory (relative standard deviation, <5.0%). The method was successfully applied to the measurement of the salivary EPA/AA ratios of healthy Japanese subjects and their changes owing to the supplementation of EPA.

LC/MS/MS of steroids having vicinal diol as electrospray-active boronates.

A derivatization procedure with (3-dimethylaminophenyl)dihydroxyborane (DAPB) was introduced to enhance the detectability of steroids having a vicinal diol in LC/electrospray ionization (ESI)-MS/MS. DAPB reacted with the vicinal diol on the steroids [4ß-hydroxycholesterol (4-HCh), pregnanetriol (PT) and 20R,22R-dihydroxycholesterol] in pyridine at 50°C within 1 h. The resulting DAPB-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using a selected reaction monitoring mode; the detection responses of the DAPB-derivatives were increased by 20-160-fold over those of the intact steroids and the limits of detection were in the low femtomole or attomole range. The derivatization procedure was successfully applied to biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the specific detection of trace amounts of 4-HCh in human plasma and PT in human urine with a small sample volume, simple pretreatment and short chromatographic run time.

Predominant role of type 1 IP3 receptor in aortic vascular muscle contraction.

Inositol 1,4,5-trisphosphate receptor (IP(3)R) plays a crucial role in generating Ca(2+) signaling and three subtypes of IP(3)R have been identified. In spite of a high degree of similarity among these subtypes, their effects on spatio-temporal Ca(2+) patterns are specific and diverse; therefore the physiological significance of the differential expression levels of IP(3)R subtypes in various tissues remains unknown. Here, we examined the relative contribution of the specific subtype of IP(3)Rs to the agonist-induced Ca(2+) signaling and contraction in IP(3)R-deficient vascular smooth muscle cells and found that IP(3)R1 deficient cells exclusively showed less sensitivity to the agonist, compared to those from the other genotypes. We also found that IP(3)R1 dominantly expressed in vascular aortae on a consistent basis, and that phenylephrine (PE)-induced aortic muscle contraction was reduced specifically in IP(3)R1-deficient aortae. Taken together, we concluded that IP(3)R1 plays a predominant role in the function of the vascular smooth muscle in vivo.

In situ visualization of caspase-1-like activity associated with promotion of hippocampal cell death.

To clarify the function of caspase-1-like proteases in neuronal cell death, it is important to be able to detect the activity in living organs by microscopic visualization. In the present study, we synthesized a novel fluorescent substrate sensitive to the caspase-1-like activity, which is easily introduced into cells constituting living organs by extracellular application. As a result, the substrate was shown to be useful in imaging the caspase-1-like activity in rat hippocampal slice cultures. After induction of cell death with glutamate, a significant increase in the activity was observed, especially in the pyramidal cells, suggesting the association of the activity with promotion of cell death.

Effect of C3 transferase on human adipose-derived stem cells.

Human adipose-derived stem cells (ASCs) are adult pluripotent stem cells, which have the ability to differentiate into fat, cartilage, bone, or nerves that can be applied in tissue engineering. On the other hand, the exoenzyme C3 transferase (C3) is a Rho inhibitor. Once in the cytosol, the cell-penetrating moiety is released, thereby allowing C3 transferase to freely diffuse intracellularly and inactivate RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1. In this study, we investigated ASC cytoskeletal changes induced by the addition of C3 employing immunofluorescence staining, changes in alpha-smooth muscle actin (a-SMA) gene expression employing real-time RT-PCR, and the Rho-inhibitory effect employing the pull-down assay. C3 significantly reduced stress fiber disruption and a-SMA expression 24 h after its addition at a concentration of 1 µg/ml, and it also reduced the Rho activity level. While the correlation of the occurrence can be assumed, it requires further examination to verify it. C3 may be an effective inhibitor of intracellular signal transmission in ASC cytoskeletal control involving Rho.

Two novel 2-aminoethyl diphenylborinate (2-APB) analogues differentially activate and inhibit store-operated Ca(2+) entry via STIM proteins.

Store-operated calcium entry (SOCE) or calcium release-activated calcium current (I(CRAC)) is a critical pathway to replenish intracellular calcium stores, and plays indispensable roles in cellular functions such as antigen-induced T lymphocyte activation. Despite the importance of I(CRAC) in cellular functions, lack of potent and specific inhibitor has limited the approaches to the function of I(CRAC) in native cells. 2-Aminoethyl diphenylborinate (2-APB) is a widely used SOCE/I(CRAC) inhibitor, while its effect is rather unspecific. In the attempt to develop more potent and selective compounds here we identified two structurally isomeric 2-APB analogues that are 100-fold more potent than 2-APB itself. One of the 2-APB analogues activates and inhibits endogenous SOCE depending on the concentration while the other only inhibits it. The 2-APB analogue inhibits store depletion-mediated STIM1 clustering as well as heterologously expressed CRAC current. Together with the observation that, unlike 2-APB, the analogue compounds failed to activate CRACM3/Orai3 current in the absence of STIM, our results suggest that inhibition and activation of SOCE/I(CRAC) by the 2-APB analogues is mediated by STIM.

Up-regulated PAR-2-mediated salivary secretion in mice deficient in muscarinic acetylcholine receptor subtypes.

Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.

Ca2+-dependent induction of intracellular Ca2+ oscillation in hippocampal astrocytes during metabotropic glutamate receptor activation.

We have investigated whether the intracellular calcium concentration ([Ca(2+)](i)) oscillations induced in astrocytes using the metabotropic glutamate-receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) are Ca(2+)-dependent, using three different Ca(2+) indicators with different affinities for Ca(2+). When rat hippocampal cells in culture were loaded with fura-2 (K(d): 145 nM), two-thirds of the cells showed obvious oscillatory increase in [Ca (2+)](i) during t-ACPD-administration. Those cells were identified as astrocytes by immuno-histochemistry in our previous paper. In cells loaded with fura-2FF (K(d): 25,000 nM), a similar percentage of t-ACPD-responsive cells showed oscillatory [Ca(2+)](i) changes. However, in cells loaded with quin-2 (K(d): 60 nM), t-ACPD induced no oscillatory responses, but some cells showed a small transient increase in the [Ca(2+)](i). The same small transient [Ca(2+)](i) increase was seen in cells loaded with both fura-2FF and BAPTA, a Ca(2+) chelator (K(d): 135 nM). These findings indicate the involvement of [Ca(2+)](i)-dependent regulatory mechanisms in the induction of the t-ACPD-induced oscillatory change in the [Ca(2+)](i) in astrocytes.

Expression of group I metabotropic glutamate receptors in rat hippocampal cells in culture and their characterization by intracellular calcium ion dynamics.

The distribution of group I metabotropic glutamate receptors in rat hippocampal cells in culture was examined by calcium imaging and immunocytochemistry. To distinguish different cell types in the culture, the effects of t-ACPD ((1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid) and of NMDA (N-methyl-D-aspartate) were examined. About 40% of the cultured cells showed either a transient increase or a sustained or oscillatory increase in the intracellular calcium concentration ([Ca(2+)](i)) during t-ACPD administration, while about 60% of the cells showed a sustained [Ca(2+)](i) increase in response to NMDA. Cells that showed an oscillatory [Ca(2+)](i) change during t-ACPD administration did not respond to NMDA administration, while cells that showed a sustained [Ca(2+)](i) increase during NMDA administration did not show any oscillatory response to t-ACPD. After pharmacological examination using those two agonists, the cultured cells were subjected to immunocytochemistry using anti-GFAP and ant-MAP-2 antibodies to distinguish, respectively, astrocytes and neurons. All cells responding to NMDA with a sustained [Ca(2+)](i) increase were MAP-2-positive, whereas all cells showing either oscillatory or sustained [Ca(2+)](i) increase in response to t-ACPD were GFAP-positive. The present results show that, in these cultures, group I metabotropic glutamate receptors are mainly expressed on glial cells and contribute to dynamic [Ca(2+)](i) changes in astrocytes.

M(3) muscarinic acetylcholine receptor plays a critical role in parasympathetic control of salivation in mice.

The M(1) and M(3) subtypes are the major muscarinic acetylcholine receptors in the salivary gland and M(3) is reported to be more abundant. However, despite initial reports of salivation abnormalities in M(3)-knockout (M(3)KO) mice, it is still unclear which subtype is functionally relevant in physiological salivation. In the present study, salivary secretory function was examined using mice lacking specific subtype(s) of muscarinic receptor. The carbachol-induced [Ca(2+)](i) increase was markedly impaired in submandibular gland cells from M(3)KO mice and completely absent in those from M(1)/M(3)KO mice. This demonstrates that M(3) and M(1) play major and minor roles, respectively, in the cholinergically induced [Ca(2+)](i) increase. Two-dimensional Ca(2+)-imaging analysis revealed the patchy distribution of M(1) in submandibular gland acini, in contrast to the ubiquitous distribution of M(3). In vivo administration of a high dose of pilocarpine (10 mg kg(-1), s.c.) to M(3)KO mice caused salivation comparable to that in wild-type mice, while no salivation was induced in M(1)/M(3)KO mice, indicating that salivation in M(3)KO mice is caused by an M(1)-mediated [Ca(2+)](i) increase. In contrast, a lower dose of pilocarpine (1 mg kg(-1), s.c.) failed to induce salivation in M(3)KO mice, but induced abundant salivation in wild-type mice, indicating that M(3)-mediated salivation has a lower threshold than M(1)-mediated salivation. In addition, M(3)KO mice, but not M(1)KO mice, had difficulty in eating dry food, as shown by frequent drinking during feeding, suggesting that salivation during eating is mediated by M(3) and that M(1) plays no practical role in it. These results show that the M(3) subtype is essential for parasympathetic control of salivation and a reasonable target for the drug treatment and gene therapy of xerostomia, including Sjögren's syndrome.