Exposure to Asian dust within a few days of delivery is associated with placental abruption in Japan: a case-crossover study.

OBJECTIVE: Asian dust is a natural phenomenon in which dust particles are transported from desert areas in China and Mongolia to East Asia. Short-term exposure to Asian dust has been associated with cardiovascular disease through mechanisms such as systemic inflammation. Because inflammation is a potential trigger of placental abruption, exposure may also lead to abruption. We examined whether exposure to Asian dust was associated with abruption. DESIGN: A bi-directional, time-stratified case-crossover design. SETTING AND POPULATION: From the Japan Perinatal Registry Network database, we identified 3014 patients who delivered singleton births in hospitals in nine Japanese prefectures from 2009 to 2014 with a diagnosis of placental abruption. METHODS: Asian dust levels were measured at Light Detection and Ranging monitoring stations, and these measurements were used to define the Asian dust days. As there was no information on the onset day of abruption, we assumed this day was the day before delivery (lag1). MAIN OUTCOME MEASURES: Placental abruption. RESULTS: During the study period, the Asian dust days ranged from 15 to 71 days, depending on the prefecture. The adjusted odds ratio of placental abruption associated with exposure to Asian dust was 1.4 (95% confidence interval = 1.0, 2.0) for cumulative lags of 1-2 days. Even after adjustment for co-pollutant exposures, this association did not change substantially. CONCLUSIONS: In this Japanese multi-area study, exposure to Asian dust was associated with an increased risk of placental abruption. TWEETABLE ABSTRACT: Exposure to environmental factors such as Asian dust may be a trigger of placental abruption.

Schizophrenia-like phenotypes in mice with NMDA receptor ablation in intralaminar thalamic nucleus cells and gene therapy-based reversal in adults.

In understanding the mechanism of schizophrenia pathogenesis, a significant finding is that drug abuse of phencyclidine or its analog ketamine causes symptoms similar to schizophrenia. Such drug effects are triggered even by administration at post-adolescent stages. Both drugs are N-methyl-d-aspartate receptor (NMDAR) antagonists, leading to a major hypothesis that glutamate hypofunction underlies schizophrenia pathogenesis. The precise region that depends on NMDAR function, however, is unclear. Here, we developed a mouse strain in which NMDARs in the intralaminar thalamic nuclei (ILN) were selectively disrupted. The mutant mice exhibited various schizophrenia-like phenotypes, including deficits in working memory, long-term spatial memory, and attention, as well as impulsivity, impaired prepulse inhibition, hyperlocomotion and hyperarousal. The electroencephalography analysis revealed that the mutant mice had a significantly reduced power in a wide range of frequencies including the alpha, beta and gamma bands, both during wake and rapid eye movement (REM) sleep, and a modest decrease of gamma power during non-REM sleep. Notably, restoring NMDARs in the adult ILN rescued some of the behavioral abnormalities. These findings suggest that NMDAR dysfunction in the ILN contributes to the pathophysiology of schizophrenia-related disorders. Furthermore, the reversal of inherent schizophrenia-like phenotypes in the adult mutant mice supports that ILN is a potential target site for a therapeutic strategy.

Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor.

The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Our previous studies have demonstrated that calmodulin binds to IP3R type I (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k(a) = 4.46 x 10(2) and > 10(4) M(-1) s(-1) k(d) = 1.44 x 10(-2) and 1.17 x 10(-1) s(-1). The apparent dissociation constant was calculated to be 27.3 microM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+.

Developmental neurotoxicity of phenytoin on granule cells and Purkinje cells in mouse cerebellum.

Phenytoin (PHT) is a primary antiepileptic drug. Cerebellar malformations in human neonates have been described following intrauterine exposure to PHT. The neonatal period of development in the cerebellum in mice corresponds to the last trimester in humans. To examine the neurotoxic effects of PHT in the developing cerebellum, we administered PHT orally to newborn mice once a day during postnatal days 2-4. We observed many apoptotic cells in the external granular layer (EGL) on postnatal day 5, labeled cells in the EGL still remaining 72 h after labeling with 5-bromo-2'-deoxyuridine, and EGL thicker than that in the control on postnatal day 14. These results showed that PHT induced cell death of external granule cells and inhibited migration of granule cells in cerebella. In specimens immunostained with antibody against inositol 1,4,5-trisphosphate receptor type 1, Purkinje cells in the treated group had poor and immature arbors, and partially showed an irregular arrangement. The motor performance of the treated mice in a rotating rod test was impaired, although there were no changes in muscular strength or in walking pattern at the period of maturity. These findings indicate that PHT induces neurotoxic damage to granule cells and Purkinje cells in the developing cerebellum and impairs selected aspects of motor coordination ability.

Trypsinized cerebellar inositol 1,4,5-trisphosphate receptor. Structural and functional coupling of cleaved ligand binding and channel domains.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a tetrameric intracellular inositol 1,4,5-trisphosphate (IP3)-gated Ca2+ release channel (calculated molecular mass = approximately 313 kDa/subunit). We studied structural and functional coupling in this protein complex by limited (controlled) trypsinization of membrane fractions from mouse cerebellum, the predominant site for IP3R1. Mouse IP3R1 (mIP3R1) was trypsinized into five major fragments (I-V) that were positioned on the entire mIP3R1 sequence by immuno-probing with 11 site-specific antibodies and by micro-sequencing of the N termini. Four fragments I-IV were derived from the N-terminal cytoplasmic region where the IP3-binding region extended over two fragments I (40/37 kDa) and II (64 kDa). The C-terminal fragment V (91 kDa) included the membrane-spanning channel region. All five fragments were pelleted by centrifugation as were membrane proteins. Furthermore, after solubilizing with 1% Triton X-100, all were co-immunoprecipitated with the C terminus-specific monoclonal antibody that recognized only the fragment V. These data suggested that the native mIP3R1-channel is an assembly of four subunits, each of which is constituted by non-covalent interactions of five major, well folded structural components I-V that are not susceptible to attack by mild trypsinolysis. Ca2+ release experiments further revealed that even the completely fragmented mIP3R1 retained significant IP3-induced Ca2+ release activity. These data suggest that structural coupling among five split components conducts functional coupling for IP3-induced Ca2+ release, despite the loss of peptide linkages. We propose structural-functional coupling in the mIP3R1, that is neighboring coupling between components I and II for IP3 binding and long-distant coupling between the IP3 binding region and the channel region (component V) beyond trypsinized gaps for ligand gating.

Cooperative formation of the ligand-binding site of the inositol 1,4, 5-trisphosphate receptor by two separable domains.

Limited trypsin digestion of mouse cerebellar membrane fractions leads to fragmentation of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) into five major components (Yoshikawa, F., Iwasaki, H., Michikawa, T., Furuichi, T., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 316-327). Here we report that trypsin-fragmented mouse IP3R1 (mIP3R1) retains significant inositol 1,4,5-trisphosphate (IP3) binding activity that is comparable to the intact receptor in affinity, capacity, and specificity. This is despite the fact that the IP3-binding core (residues 226-578), which is close to the minimum for high affinity binding, is completely split into two tryptic fragments at the Arg-343 and/or Arg-345, around the center of the core. Furthermore, we have examined whether binding activity could be complemented in vitro by mixing two distinct glutathione S-transferase (GST) fusion proteins, which were respectively composed of residues 1-343 and 341-604, almost corresponding to two split binding components, and separately expressed in Escherichia coli. The GST-fused residues 1-343 (GN) showed no binding affinity for IP3, whereas the GST-fused residues 341-604 (GC) displayed weak but definite activity with an affinity >100-fold lower than that of the native receptor. Upon mixing of both GN and GC, a high affinity site comparable to the native site appeared. We suggest that the IP3-binding pocket consists of two non-covalently but tightly associated structural domains each of which has a discrete function: the C-terminal domain alone has low affinity for IP3, whereas the N-terminal one alone is incapable of binding but is capable of potentiating binding affinity.

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha-thrombin.

The effect of inositol 1,4,5-trisphosphate (IP3) receptor blockade on platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), endothelin-1 (ET-1), or alpha-thrombin receptor-mediated intracellular Ca2+ (Ca2+i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells and myoblasts. Blockade of the IP3 receptor was achieved by microinjection of heparin or monoclonal antibody (MAb) 18A10 into the IP3 type 1 receptor. Heparin completely inhibited Ca2+i release after flash photolysis with caged IP3 and after exposure to PDGF and FGF. In contrast, heparin failed to block Ca2+i release after alpha-thrombin and ET-1. After application of ligand, IP3 levels were five- to sevenfold higher for alpha-thrombin than for ET-1 or PDGF. IP3 levels after PDGF and ET-1 were comparable. Similar to heparin, MAb 18A10 blocked Ca2+i release after PDGF but failed to block Ca2+i release after ET-1 or alpha-thrombin. These data suggest that the mechanisms of Ca2+i release by tyrosine kinase and certain 7-transmembrane receptors may differ. Although both receptor types use the IP3-signaling system, the ET-1 and alpha-thrombin receptors may have a second, alternative mechanism for activating CA2+i release.

Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin.

The N-methyl-D-aspartate (NMDA) receptor plays important roles in synaptic plasticity and brain development. The NMDA receptor subunits have large intracellular domains in the COOH-terminal region that may interact with signal-transducing proteins. By using the yeast two-hybrid system, we found that calmodulin interacts with the COOH terminus of the NR1 subunit and inactivates the channels in a Ca2+-dependent manner. Here we show that protein kinase C (PKC)-mediated phosphorylation on serine residues of NR1 decreases its affinity for calmodulin. This suggests that PKC-mediated phosphorylation of NR1 prevents calmodulin from binding to the NR1 subunit and thereby inhibits the inactivation of NMDA receptors by calmodulin. In addition, we show that stimulation of metabotropic glutamate receptor 1alpha, which potentiates NMDA channels through PKC, decreases the ability of NR1 to bind to calmodulin. Thus, our data provide clues to understanding the basis of cross-talk between two types of receptors, metabotropic glutamate receptors and the NR1 subunit, in NMDA channel potentiation.

Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.

In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.

Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors.

To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.

Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy.

We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.

Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.

To define the structural determinants for inositol 1,4, 5-trisphosphate (IP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.

Inositol 1,4,5-trisphosphate receptors and calcium signaling.

Many cellular responses to extracellular stimuli are mediated by the second messenger inositol 1,4,5-trisphosphate (InsP3). InsP3 releases Ca2+ from intracellular stores by binding to an InsP3 receptor (InSP3R), which is an InsP3-gated Ca2+ release channel. The resultant increase in the cytoplasmic Ca2+ concentration modulates various cellular functions, such as gene expression, metabolism, proliferation, secretion, and neural excitation. In these signaling cascades, InsP3R works as a signal converter from InsP3 to Ca2+. We describe here structural and functional properties and localization of InsP3R, a key molecule in the Ca2+ signaling pathway.

Kinetics of calcium release by immunoaffinity-purified inositol 1,4,5-trisphosphate receptor in reconstituted lipid vesicles.

The kinetics of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release of the immunoaffinity-purified IP3 receptor (IP3R), reconstituted into lipid vesicles, was investigated using the fluorescent Ca2+ indicator fluo-3. IP3R was purified from mouse cerebellar microsomal fraction by using an immunoaffinity column conjugated with an anti-IP3R type 1 (IP3R1) antibody. The immunoblotting analysis using monoclonal antibodies against each IP3R type showed that the purified IP3R is almost homogeneous, composed of IP3R1. Ca2+ efflux from the proteoliposomes was monitored as fluorescence changes of 10 microM fluo-3, whose concentration was high enough to buffer released Ca2+ and to keep deviations of extravesicular free Ca2+ concentration within 30 nM, excluding the possibility of Ca(2+)-mediated regulation of IP3-induced Ca2+ release. We also examined IP3-induced Ca2+ release using 1 microM fluo-3, where the deviations of free Ca2+ concentration were within 300 nM. At both fluo-3 concentrations, IP3-induced Ca2+ release showed similar kinetic properties, i.e. little Ca2+ regulation of Ca2+ release was observed in this system. IP3-induced Ca2+ release of the purified IP3R exhibited positive cooperativity; the Hill coefficient was 1.8 +/- 0.1. The half-maximal initial rate for Ca2+ release occurred at 100 nM IP3. At the submaximal concentrations of IP3, the purified IP3R showed quantal Ca2+ release, indicating that a single type of IP3R is capable of producing the phenomenon of quantal Ca2+ of release. The profiles of the IP3-induced Ca2+ release of the purified IP3R were found to be biexponential with the fast and slow rate constants (k(fast) = 0.3 approximately 0.7 s-1, k(slow) = 0.03 approximately 0.07 s-1), indicating that IP3R has two states to release CA2+. The amount of released Ca2+ by the slow phase was constant over the range of 10-5000 nM IP3 concentrations, whereas that by the fast phase increased in proportion to added IP3. This provides evidence to support the view that the fast phase of Ca2+ release is mediated by the low affinity state and the slow phase by the high affinity state of the IP3R. This also suggests that the fast component of Ca2+ release is responsible for the process of quantal Ca2+ release.

Adenophostin-medicated quantal Ca2+ release in the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Kinetics of Ca2+ release by adenophostin, a novel agonist of inositol 1,4,5-trisphosphate (IP3) receptor, in the purified and reconstituted IP3 receptor type 1 (IP3R1) was investigated using the fluorescent Ca2+ indicator fluo-3. Submaximal concentrations of adenophostin caused quantal Ca2+ release from the purified IP3R1 as IP3 did. Adenophostin-induced Ca2+ release by the purified IP3R1 exhibited a high positive cooperativity (nH = 3.9 +/- 0.2, EC50 = 11 nM), whereas the IP3-induced Ca2+ release exhibited a moderate one (nH = 1.8 +/- 0.1, EC50 = 100 nM). Inhibition of [3H]IP3 binding to the purified IP3R1 by adenophostin exhibited a positive cooperativity (nH = 1.9, Ki = 10 nM), whereas IP3 did not (nH = 1.1, Ki = 41 nM).

Transmembrane topology and sites of N-glycosylation of inositol 1,4,5-trisphosphate receptor.

To define the transmembrane topology of the inositol 1,4,5-trisphosphate receptor (InsP3R), we determined the subcellular location of the hydrophilic segment (residues 2463-2529 of mouse type 1 InsP3R) believed to be located at the luminal side of the endoplasmic reticulum (ER) in the six-transmembrane model but at the cytoplasmic side in the eight-transmembrane model. This hydrophilic segment includes two consensus sites for N-glycosylation (Asn-2475 and Asn-2503). We prepared an anti-peptide antibody against residues 2504-2523. Electron microscope immunocytochemical studies of mouse cerebellar Purkinje cells showed that binding of this antibody frequently occurs in the intracisternal space of the ER. We constructed three mutant receptors by site-directed mutagenesis of Asn to Gln (N2475Q, N2503Q, and N2475Q/N2503Q). By concanavalin A column chromatography of these receptors, we found that both Asn-2475 and Asn-2503 are glycosylated. These results indicate that residues 2504-2523, Asn-2475, and Asn-2503 are exposed to the ER lumen. We therefore propose that InsP3R has six membrane-spanning segments. Based on the transmembrane topology and subunit organization, we suggest that InsP3R is a member of the superfamily that includes the voltage- and second messenger-gated ion channels on the plasma membrane.