IP3R1 Gene Research Articles

A C1976Y missense mutation in the mouse Ip3r1 gene leads to short-term mydriasis and unfolded protein response in the iris constrictor muscles.

Ip3r1 encodes an inositol 1,4,5-trisphosphate-responsive calcium channel. Mutations in the IP3R1 gene in humans may cause Gillespie syndrome (GS) typically presents as fixed dilated pupils in affected infants, which was referred to as iris hypoplasia. However, there is no report of mice with Ip3r1 heterozygous mutations showing dilated pupils. Here, we report a new Ip3r1 allele with short-term dilated pupil phenotype derived from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. This allele carries a G5927A transition mutation in Ip3r1 gene (NM_010585), which is predicted to result in a C1976Y amino acid change in the open reading frame of IP3R1 (NP_034715). We named this novel Ip3r1 allele Ip3r1C1976Y. Histology and pharmacological tests show that the dilated pupil phenotype is a mydriasis caused by the functional defect in the iris constrictor muscles in Ip3r1C1976Y. The dilated pupil phenotype in Ip3r1C1976Y was referred to as mydriasis and excluding iris hypoplasia. IHC analysis revealed increased expression of BIP protein, the master regulator of unfolded protein response (UPR) signaling, in Ip3r1C1976Y mice that did not recover. This study is the first report of an Ip3r1 mutation being associated with the mydriasis phenotype. Ip3r1C1976Y mice represent a self-healing model that may be used to study the therapeutic approach for Ip3r1-related diseases.

Effect of Docosahexaenoic Acid on Ca2+ Signaling Pathways in Cerulein-Treated Pancreatic Acinar Cells, Determined by RNA-Sequencing Analysis.

Intracellular Ca2+ homeostasis is commonly disrupted in acute pancreatitis. Sustained Ca2+ release from internal stores in pancreatic acinar cells (PACs), mediated by inositol triphosphate receptor (IP3R) and the ryanodine receptor (RyR), plays a key role in the initiation and propagation of acute pancreatitis. Pancreatitis induced by cerulein, an analogue of cholecystokinin, causes premature activation of digestive enzymes and enhanced accumulation of cytokines and Ca2+ in the pancreas and, as such, it is a good model of acute pancreatitis. High concentrations of the omega-3 fatty acid docosahexaenoic acid (DHA) inhibit inflammatory signaling pathways and cytokine expression in PACs treated with cerulein. In the present study, we determined the effect of DHA on key regulators of Ca2+ signaling in cerulein-treated pancreatic acinar AR42 J cells. The results of RNA-Sequencing (RNA-Seq) analysis showed that cerulein up-regulates the expression of IP3R1 and RyR2 genes, and that pretreatment with DHA blocks these effects. The results of real-time PCR confirmed that DHA inhibits cerulein-induced IP3R1 and RyR2 gene expression, and demonstrated that DHA pre-treatment decreases the expression of the Relb gene, which encodes a component of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional activator complex, and the c-fos gene, which encodes a component of activator protein-1 (AP-1) transcriptional activator complex. Taken together, DHA inhibits mRNA expression of IP3R1, RyR2, Relb, and c-fos, which is related to Ca2+ network in cerulein-stimulated PACs.

Asthma-Alleviating Potential of 6-Gingerol: Effect on Cytokines, Related mRNA and c-Myc, and NFAT1 Expression in Ovalbumin-Sensitized Asthma in Rats.

In this study, we aimed at assessing the therapeutical potential of 6-gingerol ([5S]-5-hydroxy-1-[4-hydroxy- 3-methoxyphenyl]-3-decanone) against ovalbumin-sensitized asthma in rats. The rats were treated intraperitoneally with 6-gingerol (75 mg/kg body weight) for 30 days and a theophylline (200 mg/kg body weight)-treated group as a control. Changes in the levels of T-cell-linked cytokines (interleukin-4 [IL-4], IL-5, IL-13, and interferon-gamma [IFN-?]), total immunoglobulin E (IgE), gene expressions of bitter taste-sensing type 2-receptor 10 (T2R10), inositol 1,4,5-triphosphate receptor 1 (IP3R1), Orai1 and protein expressions of nuclear factor of activated T cells 1 (NFAT1), c-Myc and histopathological changes were observed in rats. 6-Gingerol exerts its beneficial impacts like theophylline in lessening IL-4, IL-5, and IL-13, and IgE and increasing the level of IFN-?. Significant down-regulation of T2R10 gene expression and up-regulation of IP3R1 and Orai1 gene expression were observed in experimental rats and these alterations were normalized after treatment with 6-gingerol or theophylline. The histopathological study revealed that the accumulation of glycoprotein and thickness of alveolar epithelium in asthmatic rats and supplementation with 6-gingerol or theophylline in asthmatic rats restored these changes to normal. In conclusion, 6-gingerol has a protective effect on lungs in ovalbumin-sensitized asthma in rats.

Inositol 1,4,5-triphosphate receptor is selectively expressed in cerebellum but not cerebellum-like structures of the elasmobranch fish, Leucoraja erinacea

The Inositol 1,4,5-trisphosphate receptor type 1 protein (Ip3r1) performs an essential role for the induction of cerebellar long-term depression. Here, I describe the use of RT-PCR, qPCR, western blotting and immunohistochemistry to assay Ip3r1 gene expression and localize Ip3r1 protein in the hindbrain of the elasmobranch fish, Leucoraja erinacea. Elasmobranchs are representatives of the most basal, yet extant lineage of gnathostomes, or jawed vertebrates. The cerebellum is a synapomorphy for gnathostomes and thus elasmobranch cerebellar physiology may serve as a proxy for the ancestral state of other jawed vertebrates. LeIp3r1 is selectively expressed in the cerebellum of the little skate and the resultant protein is localized to Purkinje cells. If Ip3r1 performs the same functions in the skate cerebellum as in the mammalian cerebellum, then parallel fiber-Purkinje cell long-term depression through Ip3r1 mediated intracellular calcium regulation may be a conserved feature of cerebellar physiology. Cerebellum and surrounding hindbrain regions termed cerebellum-like structures share a common developmental genetic toolkit. LeIp3r1 expression was lowly detected in cerebellum-like structures indicating that although generatively homologous, the cerebellum and cerebellum-like structures do not share a complete overlap of common expression. Because of the little skate's important phylogenetic placement, performing molecular methodologies to assay targeted gene expression and determine protein localization in the hindbrain can be valuable for our understanding of cerebellar evolution and comparative neural development.

Identification of novel regulatory GRE-binding elements in the porcine IP3R1 gene promoter and their transcriptional activation under glucocorticoid stimulation

Inositol 1,4,5-trisphosphate receptor 1 (IP3R1) is a type of ligand-gated calcium channel that is expressed predominantly in mammalian skeletal muscle, where it acts as a key regulator of calcium homeostasis. In meat, calcium disequilibrium is accompanied by the deterioration of meat quality. Here we show that serum cortisol concentration was higher and the IP3R1 gene expression level increased markedly in pigs exposed to high stress. In porcine primary muscle cells, dexamethasone (DEX, a synthetic glucocorticoid) increased the protein levels of porcine IP3R1 and GRα, and cell apoptosis, and the specific GRα inhibitor RU486 attenuated these effects. DEX also increased the expression of IP3R1 at both the gene and protein levels, and this expression was attenuated by RU486, siRNA against GRα, and the transcriptional inhibitor actinomycin D. DEX significantly reduced cell viability and increased the intracellular calcium concentration, and these effects were attenuated by siRNA against GRα. Bioinformatics analyses predicted a potential glucocorticoid response element (GRE) located in the region −326 to −309 upstream of the IP3R1 promoter and highly conserved in pigs and other mammalian species. Promoter analysis showed that this region containing the GRE was critical for transcriptional activity of porcine IP3R1 under DEX stimulation. This was confirmed by deletion and site-mutation methods. EMSA and ChIP assays showed that this potential GRE bound specifically to GRα and this complex activated the transcription of the IP3R1 gene. Taken together, these data suggest that DEX-mediated induction of IP3R1 influences porcine muscle cells through the transcriptional activation of a mechanism involving interactions between GRα and a GRE present in the proximal IP3R1 promoter. This process can lead to an imbalance in intracellular calcium concentration, which may subsequently activate the apoptosis signal and decrease cell activity, and cause deterioration of meat quality.

Functional Role of Intracellular Calcium Receptor Inositol 1,4,5-Trisphosphate Type 1 in Rat Hippocampus after Neonatal Anoxia.

Anoxia is one of the most prevalent causes of neonatal morbidity and mortality, especially in preterm neonates, constituting an important public health problem due to permanent neurological sequelae observed in patients. Oxygen deprivation triggers a series of simultaneous cascades, culminating in cell death mainly located in more vulnerable metabolic brain regions, such as the hippocampus. In the process of cell death by oxygen deprivation, cytosolic calcium plays crucial roles. Intracellular inositol 1,4,5-trisphosphate receptors (IP3Rs) are important regulators of cytosolic calcium levels, although the role of these receptors in neonatal anoxia is completely unknown. This study focused on the functional role of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) in rat hippocampus after neonatal anoxia. Quantitative real-time PCR revealed a decrease of IP3R1 gene expression 24 hours after neonatal anoxia. We detected that IP3R1 accumulates specially in CA1, and this spatial pattern did not change after neonatal anoxia. Interestingly, we observed that anoxia triggers translocation of IP3R1 to nucleus in hippocampal cells. We were able to observe that anoxia changes distribution of IP3R1 immunofluorescence signals, as revealed by cluster size analysis. We next examined the role of IP3R1 in the neuronal cell loss triggered by neonatal anoxia. Intrahippocampal injection of non-specific IP3R1 blocker 2-APB clearly reduced the number of Fluoro-Jade C and Tunel positive cells, revealing that activation of IP3R1 increases cell death after neonatal anoxia. Finally, we aimed to disclose mechanistics of IP3R1 in cell death. We were able to determine that blockade of IP3R1 did not reduced the distribution and pixel density of activated caspase 3-positive cells, indicating that the participation of IP3R1 in neuronal cell loss is not related to classical caspase-mediated apoptosis. In summary, this study may contribute to new perspectives in the investigation of neurodegenerative mechanisms triggered by oxygen deprivation.

Selaginella uncinata flavonoids ameliorated ovalbumin-induced airway inflammation in a rat model of asthma

Ethnopharmacological relevanceSelaginella uncinata (Desv.) Spring, known as “Cuiyuncao”, is a perennial herb widely distributed in the Southeast Asian countries. In the folk medicine, the local minority commonly use it to treat cough and asthma for centuries. Aim of the studyThis study was carried out to investigate the protective mechanisms of total flavonoids from S. uncinata (SUF) on airway hyperresponsiveness, cytokine release and bitter taste receptors (T2Rs) signaling with emphasis on inflammatory responses in a rat model of ovalbumin (OVA)-induced asthma. Materials and methodsRats were sensitized and challenged with OVA to induce typical asthmatic reactions. Pathological changes of lung tissue were examined by HE staining. The serum levels of T cell-associated cytokines (IFN-γ, IL-4, IL-5 and IL-13), total IgE and OVA-specific IgE were determined by enzyme-linked immunosorbent assay (ELISA). Gene expressions of T2R10, IP3R1 and Orai1 in lung tissue were assayed by fluorescence quantitative real-time polymerase chain reaction (FQ-PCR) while protein expressions of NFAT1 and c-Myc were assayed by western blot analysis. The activation of SUF was investigated on tansgentic T2R10-GFP HEK293 cells. ResultsSUF treatment attenuated airway hyperresponsiveness and goblet cell hyperplasia compared with OVA-challenged asthmatic rats. The serum levels of IL-4, IL-5 and IL-13 as well as total and OVA-specific IgE were decreased while serum IFN-γ was increased in SUF-treated rats. SUF treatment significantly up-regulated T2R10 gene expression, down-regulated IP3R1 and Orai1 gene expression. SUF further suppressed eotaxin, NFAT1 and c-Myc protein expression in lung tissues of OVA-challenged rats. ConclusionsThese results imply that SUF exerts anti-inflammatory function through the T2R10/IP3R1/NFAT1 dependent signaling pathway, and may warrant further evaluation as a possible agent for the treatment of asthma.

Sulforaphane-induced apoptosis involves the type 1 IP3 receptor.

In this study we show that anti-tumor effect of sulforaphane (SFN) is partially realized through the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). This effect was verified in vitro on three different stable cell lines and also in vivo on the model of nude mice with developed tumors. Early response (6 hours) of A2780 ovarian carcinoma cells to SFN treatment involves generation of mitochondrial ROS and increased transcription of NRF2 and its downstream regulated genes including heme oxygenase 1, NAD(P)H:quinine oxidoreductase 1, and KLF9. Prolonged SFN treatment (24 hours) upregulated expression of NRF2 and IP3R1. SFN induces a time-dependent phosphorylation wave of HSP27. Use of IP3R inhibitor Xestospongin C (Xest) attenuates both SFN-induced apoptosis and the level of NRF2 protein expression. In addition, Xest partially attenuates anti-tumor effect of SFN in vivo. SFN-induced apoptosis is completely inhibited by silencing of IP3R1 gene but only partially blocked by silencing of NRF2; silencing of IP3R2 and IP3R3 had no effect on these cells. Xest inhibitor does not significantly modify SFN-induced increase in the rapid activity of ARE and AP1 responsive elements. We found that Xest effectively reverses the SFN-dependent increase of nuclear content and decrease of reticular calcium content. In addition, immunofluorescent staining with IP3R1 antibody revealed that SFN treatment induces translocation of IP3R1 to the nucleus. Our results clearly show that IP3R1 is involved in SFN-induced apoptosis through the depletion of reticular calcium and modulation of transcription factors through nuclear calcium up-regulation.

Regulation of type 1 IP3 receptor expression by dopamine D2-like receptors via AP-1 and NFATc4 activation

Type 1 inositol 1,4,5-trisphosphate receptors (IP3Rs-1), together with ryanodine receptors, are major calcium channels to regulate intracellular Ca2+ concentration. Although our recent report demonstrates the essential involvement of IP3R-1 up-regulation induced by dopamine D1-like and D2-like receptor (D1 and D2R) stimulation in psychological dependence, exact regulatory mechanisms of IP3R-1 expression by D2Rs have not yet been clarified. Mouse cerebral cortical neurons were treated with inhibitor of Ca2+-related signal transduction pathways coupling to D2Rs and used to analyze the mechanisms of IP3R-1 expression regulated by transcriptional factor. A selective D2R agonist, quinpirole, up-regulated IP3R-1 protein following its mRNA increase, which was significantly inhibited by gallein (a Gβγ modulator), U73122 (a phospholipase C inhibitor), BAPTA-AM (an intracellular calcium chelating reagent), W7 (a calmodulin inhibitor), KN-93 (a calmodulin-dependent protein kinases inhibitor), and FK506 (a calcineurin inhibitor). Immunocytochemical assessment showed that quinpirole increased expression of both cFos and phosphorylated-cJun in nucleus and enhanced translocation of NFATc4 complex to nucleus from cytoplasm. In addition, quinpirole directly recruited bindings between AP-1 and IP3R-1 promoter region and between NFATc4 and IP3R-1 promoter region. These results indicate that D2Rs enhance IP3R-1 gene transcription via increased bindings of AP-1 and NFATc4 to IP3R-1 promoter region after Gβγ activation.

Different roles of inositol 1,4,5-trisphosphate receptor subtypes in prostaglandin F2α-induced calcium oscillations and pacemaking activity of NRK fibroblasts

We investigated the role of inositol 1,4,5-trisphosphate (IP3)-receptor isoforms in the prostaglandin F2α (PGF2α)-induced calcium oscillations and pacemaking activity of normal rat kidney (NRK) fibroblasts. Reverse transcription polymerase chain reaction (RT-PCR) studies revealed that NRK fibroblasts express only the IP3-receptor subtypes IP3R1 and IP3R3. Quantitative RT-PCR analysis demonstrated that their expression levels varied as a function of the growth status of NRK cells; NRK cells made quiescent (Q) by serum deprivation expressed significantly higher levels of subtypes 1 and 3 than cells grown to density-arrest (DA). Using Ca2+-imaging techniques, we show that the frequency of PGF2α-induced calcium oscillations in DA-cells is lower than in Q-cells. To study whether these differences in the frequency of calcium oscillations relate to the relative amounts of IP3-receptor subtypes expressed by the cells, we knocked down the genes for either IP3-receptor subtype by using an shRNA approach. Knockdown of the IP3R1 gene significantly decreased the frequency of the PGF2α-induced calcium oscillations in both Q- and DA-cells. It also reduced the frequency of the repetitive firing of calcium action potentials by DA-cells. In contrast, knockdown of the IP3R3 gene caused an increase in the frequency of both processes, suggesting a role for this receptor subtype as an anti-Ca2+-oscillatory unit in NRK fibroblasts. Our findings indicate that the reduction in the frequency of PGF2α-induced calcium oscillations in DA-cells compared with Q-cells results from the reduced expression ratio of IP3R1 versus IP3R3 receptors in DA-cells. Moreover, these data provide direct evidence that the frequency of IP3-dependent calcium oscillations determines the periodicity of action potential firing by density-arrested NRK fibroblasts.

Altered expression of type 1 inositol 1,4,5-trisphosphate receptor in the Ngsk Prnp deficient mice

Doppel protein (Dpl) is a paralog of the cellular form of prion protein (PrP C). Its ectopic expression in the CNS elicits significant cerebellar Purkinje cell degeneration in some lines of PrP knockout mice. However, little is known about the Dpl-mediated neurodegenerative mechanism. To understand the molecular and intracellular pathways underlying Purkinje cell degeneration, here, we investigated the regulation of calcium-release channel protein, type 1 inositol 1,4,5-trisphosphate receptor (IP 3R1) gene in Ngsk mice. These knockout mice express high levels of Dpl and eventually develop cerebellar degeneration. We observed that the expression level of IP 3R1 gene is reduced in the cerebella of Ngsk mice as early as 3 months of age compared with age-matched controls along with the reduction in DNA binding activity of nuclear factor of activated-T cells (NFAT) which is transcription factor of IP 3R1. Notably, expression of PrP restored the reduced DNA binding activity of NFATc4 by Dpl. Reduced expressions of brain-derived neurotrophic factor (BDNF) and ionotropic glutamate receptor subtype 2 or B (GluR2), which are regulated by NFATc4, were also restored by PrP expression. In light of these findings, we suggest a mechanism for Dpl-mediated Purkinje cell degeneration linked to reduced gene expression of proteins related to neuronal activity. Decrease in IP 3R1 gene expression may lead to functional deficits and ultimately death of Purkinje cells in Ngsk mice.

한우 Inositol 1,4,5-triphosphate Receptor Type 1 (IP3R1) 유전자의 다형성 및 형질 관련성 분석

본 연구는 한우 inositol 1,4,5-triphosphate receptor type1(IP3R1) 유전자를 대상으로 SNP를 발굴하고, 도체형질과의 관련성 분석을 위하여 수행하였다. PCR 및 염기서열 결정법을 통해 IP3R1 유전자내 3개의 SNP를 발굴하였고, 이중 intron 29에 위치하는 SNP의 경우 미 보고된 신규 SNP로 확인되었다. 발굴된 3개의 SNP를 대상으로 표현형 기록치를 보유한 후대검정우 583두에 대하여 유전자형 분석 및 관련성 분석을 수행하였다. 분석결과 3개의 SNP 중 g.1428617A>G SNP가 생시체중(P<0.05) 및 도체중(P<0.01)과 유의적인 상관관계가 있음을 확인할 수 있었다. 이러한 결과는 추후 한우 개량을 위한 유전자 마커로 활용이 가능할 것으로 판단된다. Inositol 1,4,5-triphosphate receptor type 1 (IP3R1) is a <TEX>$Ca^{2+}$</TEX> release channel that responds to the second messenger IP3 and that modulates diverse cellular functions such as contraction/excitation, secretion, gene expression and cellular growth. We discovered single nucleotide polymorphisms (SNPs) within IP3R1 gene and analyzed associations between gene polymorphisms and carcass traits in Korean cattle (Hanwoo) in order to develop novel DNA markers at genomic level. Three SNPs were detected at the position of g.1428617A>G, g.1418843C>T and g.1414377C>T with 24 unrelated Hanwoo samples by direct sequencing of the PCR products. We found that genotype of g.1414377C>T SNP was associated with live weight (P<0.05) and carcass weight (P<0.01) using the general linear model of SAS package. These results suggest that polymorphism of IP3R1 gene was associated with weight-related traits in Hanwoo.

Hydrogen peroxide increases GABAergic mIPSC through presynaptic release of calcium from IP3 receptor‐sensitive stores in spinal cord substantia gelatinosa neurons

GABAergic interneurons of the spinal cord substantia gelatinosa regulate the transmission of nociceptive information. Hydrogen peroxide (H2O2) is likely a diffusible messenger contributing to the development of long-lasting pathological pain states after nerve injury. In this study, we examined the presynaptic effects of H2O2 on the inhibitory interneurons of mouse substantia gelatinosa (SG) using whole-cell patch-clamp recordings from spinal cord slices. H2O2 increased the frequency of GABAergic miniature inhibitory postsynaptic current (mIPSC) in a concentration-dependent (10-1000 microM) manner. The profound increase in mIPSC frequency was diminished by thapsigargin or cyclopiazonic acid suggesting that the intracellular stored pool was the source of presynaptic calcium. Further examination revealed the 2-aminoethoxydiphenil borate blockable inositol-(1,4,5) trisphosphate receptor (IP3R) regulated pool of stored calcium as the likely source. The phospholipase C (PLC) blocker, 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U73122), did not block the frequency increase, which suggested that the site of action of H2O2 lies downstream in the IP3 signalling pathway, and nifedipine-sensitivity of the frequency increase indicated a possible role of calcium-induced calcium-release. However, a direct examination of L-type voltage-gated calcium channels (VGCC) demonstrated that H2O2 did not increase the calcium influx through these channels. The H2O2 effect on mIPSC frequency was markedly reduced in the opisthotonus (Opt) mutant mice with a known deletion in the IP3R1 gene. We demonstrated that H2O2 increased presynaptic activity in the GABAergic interneurons by the release of calcium from the IP3R-regulated intracellular pool. The presynaptic IP3R could emerge as a novel target for preventing H2O2-induced synaptic plasticity in substantia gelatinosa leading to pathological pain states.

Phospholipid Scramblase 1 Binds to the Promoter Region of the Inositol 1,4,5-Triphosphate Receptor Type 1 Gene to Enhance Its Expression

Phospholipid scramblase 1 (PLSCR1) is a multiply palmitoylated, endofacial membrane protein originally identified based on its capacity to promote accelerated transbilayer phospholipid movement in response to Ca(2+). Recent evidence suggests that this protein also participates in cell response to various growth factors and cytokines, influencing myeloid differentiation, tumor growth, and the antiviral activity of interferon. Whereas plasma membrane PLSCR1 was shown to be required for normal recruitment and activation of Src kinase by stimulated cell surface growth factor receptors, PLSCR1 was also found to traffic into the nucleus and to tightly bind to genomic DNA, suggesting a possible additional nuclear function. We now report evidence that PLSCR1 directly binds to the 5'-promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene (IP3R1) to enhance expression of the receptor. Probing a CpG island genomic library with PLSCR1 as bait identified four clones with avidity for PLSCR1, including a 191-bp fragment of the IP3R1 promoter. Using electrophoretic mobility shift and transcription reporter assays, the PLSCR1-binding site in IP3R1 was mapped to residues (-101)GTAACCATGTGGA(-89), and the segment spanning Met(86)-Glu(118) in PLSCR1 was identified to mediate its transcriptional activity. The significance of this interaction between PLSCR1 and IP3R1 in situ was confirmed by comparing levels of IP3R1 mRNA and protein in matched cells that either expressed or were deficient in PLSCR1. These data suggest that in addition to its role at the plasma membrane, effects of PLSCR1 on cell proliferative and maturational responses may also relate to alterations in expression of cellular IP3 receptors.

Plasma membrane calcium ATPase overexpression in arterial smooth muscle increases vasomotor responsiveness and blood pressure.

In vascular smooth muscle cells (SMCs), several mechanisms act in concert to regulate the intracellular calcium concentration [Ca2+]i, which may in turn affect vascular tone. One such mechanism is the extrusion of Ca2+ by the plasma membrane calcium ATPase (PMCA). To address, in particular, the role of the neuronal nitric oxide synthase (nNOS)-associating isoform PMCA4b in regulating vascular tone, a doxycycline-responsive transgene for human PMCA4b was overexpressed in arterial SMCs of mice. Overexpression of hPMCA4b resulted in a 2-fold increase in total aortic PMCA4 protein expression and significant real-time RT-PCR-documented differences in the levels of endogenous mouse PMCA1, PMCA4, SERCA2, and IP3R1 gene expression in arterial SMCs. Whereas no significant difference in basal [Ca2+]i or Ca2+ sensitivity was observed in vascular SMCs or mesenteric arteries, respectively, from hPMCA4b-overexpressing versus control mice, hPMCA4b-overexpressing mice revealed a reduced set-point and increased extent of myogenic response and heightened sensitivity to vasoconstrictors. Treatment of arteries with an nNOS inhibitor resulted in a reduced set-point and increased extent of the myogenic response in control but not hPMCA4b-overexpressing mice. Moreover, aortic SMCs from hPMCA4b-overexpressing mice exhibited reduced levels of cGMP under both basal and phenylephrine-stimulated conditions. These changes were associated with significant doxycycline-reversible elevations in blood pressure. Taken together, these data show that overexpression of hPMCA4b in arterial SMCs increases vascular reactivity and blood pressure, an effect that may be mediated in part by negative regulation of nNOS.

Type 1 inositol 1,4,5-trisphosphate receptor knock-out mice: their phenotypes and their meaning in neuroscience and clinical practice.

Cytoplasmic calcium, which acts as a second messenger, is derived not only from outside the cell but also from intracellular stores. A receptor for inositol 1,4,5-trisphosphate (IP3), an intracellular second messenger, is located on these internal calcium stores and functions as a calcium releasing channel. The "type 1" IP3 receptor (IP3R1) is concentrated predominantly in cerebellar Purkinje cells and is also widely present in other neural and peripheral tissues, but many of its physiological roles in these cells are still unclear. We have previously succeeded in obtaining mice with disruption of this IP3R1 gene, in which brain IP3-induced calcium release was almost completely abolished. They were rarely born alive, indicating that IP3R1 has some functions during embryonic development. Animals exhibited severe neurological symptoms, ataxia and epilepsy, and were shown to be deficient in the cerebellar long-term depression. They give us promising clues regarding the physiological roles of calcium release from internal stores and serve as a model for the relevant human disease states.

Transcriptional regulation of mouse type 1 inositol 1,4,5-trisphosphate receptor gene by NeuroD-related factor.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box-I element, which is located -334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E-box consensus sequence, is involved in the up-regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS-related basic helix-loop-helix (bHLH) factors bind to the box-I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS-related bHLH factors, neuronal differentiation factor (NeuroD)-related factor (NDRF), specifically bound to the box-I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co-expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue-specific regulation of IP3R1 gene expression in the CNS.

Activation of the mouse inositol 1,4,5-trisphosphate receptor type 1 promoter by AP-2.

Inositol 1,4,5-trisphosphate receptor (IP3R) functions as a Ca2+ channel that increases the intracellular Ca2+ upon binding to inositol trisphosphates. IP3R is expressed ubiquitously and consists of a multigene family. Since the type 1 IP3R (IP3R1) is highly expressed in the cerebellar Purkinje cells and moderately in hippocampus in the mammalian central nervous system (CNS), it is regarded as a neural member of this gene family. In this work, we investigated transcriptional regulation of the mouse ip3r1 gene. A DNaseI footprinting assay demonstrated that a sequence from -95 to -75, designated as box-II, was a binding site for a cerebellum-enriched factor. A consensus sequence for AP-2 was located in box-II. An electrophoretic mobility shift assay with anti-AP-2 antibody revealed that AP-2 is capable of binding to box-II. Deletion analysis of box-II showed that flanking sequences beside the box-II motif were required for the stable binding. We demonstrated by transient luciferase assay that exogenously expressed AP-2 activated box-II-dependent transcription. Moreover, we showed that endogenous AP-2 induced by retinoic acid also activated transcription via box-II in P19 cells. In-situ hybridization of the mouse brain revealed that AP-2 was predominantly expressed in the cerebellar Purkinje cells and hippocampal CA1 region, where IP3R1 is also highly expressed. From these observations, AP-2 binding to box-II is thought to be responsible for IP3R1 gene regulation in the CNS.

Demonstration of an E-box and its CNS-related binding factors for transcriptional regulation of the mouse type 1 inositol 1,4,5-trisphosphate receptor gene.

The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is expressed abundantly in the CNS, such as in cerebellar Purkinje cells and the hippocampus. We established a tissue-specific cell-free transcription system and studied regulatory properties of the 5' upstream region of the IP3R1 gene by use of this system. Deletion analyses of the promoter revealed several cis elements that function significantly in brain nuclear extracts. Among those elements, sequences from -398 to -295 showed the most predominant cerebellum-specific positive function. Footprint analyses demonstrated a factor-binding region from -334 to -318, termed box-I, that contained an E-box consensus sequence. Electrophoretic mobility shift assay revealed CNS-related basic helix-loop-helix proteins for the box-I. Mutational studies using the function assay and competitive electrophoretic mobility shift assays demonstrated a good correlation between the box-I-binding factors and the activated transcription. Box-I-binding factors were present abundantly in adult mouse CNS, whereas their existence was restricted in embryonic and nonneural tissues. Transient chloramphenicol acetyltransferase assay for the IP3R1 promoter revealed the requirement of box-I in Neuro2a neuroblastoma cells. In the postnatal CNS, multiple basic helix-loop-helix factors are expressed abundantly, some of which are suggested to activate IP3R1 gene expression in the mammalian CNS.

The Type 1 Inositol 1,4,5-Trisphosphate Receptor Gene Is Altered in theopisthotonosMouse

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.