OBJECTIVE: To explore the clinical and genetic characteristics of a child with spinocerebellar ataxia type 29 (SCA29) due to novel variant of the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) gene. METHODS: The child was subjected high-throughput sequencing, and candidate variant was verified by Sanger sequencing of his family members. RESULTS: The child was found to harbor a c.800C>T (p.T267M) variant of the ITPR1 gene, which was not found in his parents and their fetus. The variant has occurred in a hotspot of the ITPR1 gene variants and was unreported before in China. Based on his clinical and genetic characteristics, the child was diagnosed with SCA29. CONCLUSION The novel heterozygous c.800C>T (p.T267M) of the ITPR1 gene probably underlay the SCA29 in this child.
Child ; Humans ; Family ; Inositol 1,4,5-Trisphosphate Receptors/genetics* ; Mutation ; Spinocerebellar Ataxias/genetics* ; Spinocerebellar Degenerations

The purpose of the present study was to determine the role of inositol 1,4,5-trisphosphate receptor 3 (IP₃R3) in renal cyst development in autosomal dominant polycystic kidney disease (ADPKD). 2-aminoethoxy-diphenyl borate (2-APB) and shRNA were used to suppress the expression of IP₃R3. The effect of IP₃R3 on cyst growth was investigated in Madin-Darby canine kidney (MDCK) cyst model, embryonic kidney cyst model and kidney specific Pkd1 knockout (PKD) mouse model. The underlying mechanism of IP₃R3 in promoting renal cyst development was investigated by Western blot and immunofluorescence staining. The results showed that the expression level of IP₃R3 was significantly increased in the kidneys of PKD mice. Inhibiting IP₃R3 by 2-APB or shRNA significantly retarded cyst expansion in MDCK cyst model and embryonic kidney cyst model. Western blot and immunofluorescence staining results showed that hyperactivated cAMP-PKA signaling pathway in the growth process of ADPKD cyst promoted the expression of IP₃R3, which was accompanied by a subcellular redistribution process in which IP₃R3 was translocated from endoplasmic reticulum to intercellular junction. The abnormal expression and subcellular localization of IP₃R3 further promoted cyst epithelial cell proliferation by activating MAPK and mTOR signaling pathways and accelerating cell cycle. These results suggest that the expression and subcellular distribution of IP₃R3 are involved in promoting renal cyst development, which implies IP₃R3 as a potential therapeutic target of ADPKD.
Animals ; Dogs ; Mice ; Cysts/genetics* ; Inositol 1,4,5-Trisphosphate Receptors/pharmacology* ; Kidney/metabolism* ; Polycystic Kidney Diseases/metabolism* ; Polycystic Kidney, Autosomal Dominant/drug therapy* ; Madin Darby Canine Kidney Cells

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an important intracellular channel for releasing Ca²⁺. In order to investigate the effects of the ITPR1 overexpression on Ca²⁺ concentration and lipid content in duck uterine epithelial cells and its effects on calcium transport-related genes, the structural domain of ITPR1 gene of duck was cloned into an eukaryotic expression vector and transfected into duck uterine epithelial cells. The overexpression of the ITPR1 gene, the concentration of Ca²⁺, the lipid content, and the expression of other 6 calcium transport-related genes was determined. The results showed that the concentration of Ca²⁺ in uterine epithelial cells was significantly reduced after transfection (P<0.05), the triglyceride content was significantly increased (P<0.01), and the high-density lipoprotein content was significantly decreased (P<0.01). The correlation analysis results showed that the overexpression of the C-terminal half of the ITPR1 gene was significantly positively correlated with the total cholesterol content (P<0.01), which was significantly positively correlated with the low-density lipoprotein content (P<0.05). The overexpression of the N-terminal half of the ITPR1 gene was significantly positively correlated with the triglyceride content (P<0.01), which was significantly negatively correlated with the concentration of Ca²⁺ (P<0.05). RT-qPCR results of 6 calcium transport-related genes showed that the overexpression of the C-terminal half of the ITPR1 gene significantly inhibited the expression of the IP3R2, VDAC2 and CAV1 genes, and the overexpression of the N-terminal half of the ITPR1 gene significantly promoted the expression of the IP3R3 and CACNA2D1 genes. In conclusion, the ITPR1 gene overexpression can promote Ca²⁺ release in duck uterus epithelial cells, promote the synthesis of triglyceride, low-density lipoprotein and cholesterol, and inhibit the production of high-density lipoprotein, and the ITPR1 gene overexpression affected the expression of all 6 calcium transport-related genes.
Animals ; Calcium/metabolism* ; Ducks/genetics* ; Epithelial Cells ; Female ; Inositol ; Inositol 1,4,5-Trisphosphate Receptors ; Lipids ; Uterus

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER^® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER^® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER^® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.
Acrosome Reaction/physiology* ; Calcimycin/pharmacology* ; Calcium/pharmacology* ; Calcium Ionophores/pharmacology* ; Drug Delivery Systems ; Humans ; Inositol 1,4,5-Trisphosphate Receptors/metabolism* ; Male ; Progesterone/pharmacology* ; Spermatozoa/metabolism* ; Zona Pellucida/metabolism*

Intracellular Ca²⁺ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.
Acinar Cells* ; Animals ; Antioxidants ; Calcium ; Catalase ; Cell Membrane* ; Cytosol ; Dithiothreitol ; Extracellular Fluid ; Hand ; Hydrogen Peroxide* ; Hydrogen* ; Inositol 1,4,5-Trisphosphate ; Ions ; Manganese ; Mice* ; Perfusion ; Plasma Membrane Calcium-Transporting ATPases ; Plasma* ; Reactive Oxygen Species

Radial glial cells (RGCs) which function as neural stem cells are known to be non-excitable and their proliferation depends on the intracellular calcium (Ca²⁺) level. It has been well established that Inositol 1,4,5-trisphosphate (IP3)-mediated Ca²⁺ release and Ca²⁺ entry through various Ca²⁺ channels are involved in the proliferation of RGCs. Furthermore, RGCs line the ventricular wall and are exposed to a shear stress due to a physical contact with the cerebrospinal fluid (CSF). However, little is known about how the Ca²⁺ entry through mechanosensitive ion channels affects the proliferation of RGCs. Hence, we hypothesized that shear stress due to a flow of CSF boosts the proliferative potential of RGCs possibly via an activation of mechanosensitive Ca²⁺ channel during the embryonic brain development. Here, we developed a new microfluidic two-dimensional culture system to establish a link between the flow shear stress and the proliferative activity of cultured RGCs. Using this microfluidic device, we successfully visualized the artificial CSF and RGCs in direct contact and found a significant enhancement of proliferative capacity of RGCs in response to increased shear stress. To determine if there are any mechanosensitive ion channels involved, a mechanical stimulation by poking was given to individual RGCs. We found that a poking on radial glial cell induced an increase in intracellular Ca²⁺ level, which disappeared under the extracellular Ca²⁺-free condition. Our results suggest that the shear stress by CSF flow possibly activates mechanosensitive Ca²⁺ channels, which gives rise to a Ca²⁺ entry which enhances the proliferative capacity of RGCs.
Brain ; Calcium Channels* ; Calcium* ; Cerebrospinal Fluid ; Ependymoglial Cells* ; Inositol 1,4,5-Trisphosphate ; Ion Channels ; Lab-On-A-Chip Devices ; Microfluidics ; Neural Stem Cells

Ginseng gintonin is an exogenous ligand of lysophosphatidic acid (LPA) receptors. Accumulating evidence shows LPA helps in rapid recovery of corneal damage. The aim of this study was to evaluate the therapeutic efficacy of gintonin in a rabbit model of corneal damage. We investigated the signal transduction pathway of gintonin in human corneal epithelium (HCE) cells to elucidate the underlying molecular mechanism. We next evaluated the therapeutic effects of gintonin, using a rabbit model of corneal damage, by undertaking histochemical analysis. Treatment of gintonin to HCE cells induced transient increases of [Ca²⁺](i) in concentration-dependent and reversible manners. Gintonin-mediated mobilization of [Ca²⁺](i) was attenuated by LPA1/3 receptor antagonist Ki16425, phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca²⁺ chelator BAPTA-AM. Gintonin facilitated in vitro wound healing in a concentration-dependent manner. When applied as an eye-drop to rabbits with corneal damage, gintonin rapidly promoted recovery. Histochemical analysis showed gintonin decreased corneal apoptosis and increased corneal cell proliferation. We demonstrated that LPA receptor activation by gintonin is linked to in vitro and in vivo therapeutic effects against corneal damage. Gintonin can be applied as a clinical agent for the rapid healing of corneal damage.
Apoptosis ; Cell Proliferation ; Corneal Injuries ; Epithelium, Corneal ; Humans ; In Vitro Techniques ; Inositol 1,4,5-Trisphosphate ; Mortuary Practice ; Panax ; Rabbits ; Receptors, Lysophosphatidic Acid ; Signal Transduction ; Therapeutic Uses ; Type C Phospholipases ; Wound Healing* ; Wounds and Injuries*

Calcium has versatile roles in diverse physiological functions. Among these functions, intracellular Ca²⁺ plays a key role during the secretion of salivary glands. In this review, we introduce the diverse cellular components involved in the saliva secretion and related dynamic intracellular Ca²⁺ signals. Calcium acts as a critical second messenger for channel activation, protein translocation, and volume regulation, which are essential events for achieving the salivary secretion. In the secretory process, Ca²⁺ activates K⁺ and Cl⁻ channels to transport water and electrolyte constituting whole saliva. We also focus on the Ca²⁺ signals from intracellular stores with discussion about detailed molecular mechanism underlying the generation of characteristic Ca²⁺ patterns. In particular, inositol triphosphate signal is a main trigger for inducing Ca²⁺ signals required for the salivary gland functions. The biphasic response of inositol triphosphate receptor and Ca²⁺ pumps generate a self-limiting pattern of Ca²⁺ efflux, resulting in Ca²⁺ oscillations. The regenerative Ca²⁺ oscillations have been detected in salivary gland cells, but the exact mechanism and function of the signals need to be elucidated. In future, we expect that further investigations will be performed toward better understanding of the spatiotemporal role of Ca²⁺ signals in regulating salivary secretion.
Calcium Signaling ; Calcium ; Chloride Channels ; Inositol ; Inositol 1,4,5-Trisphosphate Receptors ; Protein Transport ; Saliva ; Salivary Glands ; Salivation ; Second Messenger Systems ; Secretory Pathway ; Water

OBJECTIVETo investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP).

METHODSTwenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i.

RESULTSThe pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 μg/mL) and IP3 (872.2 ± 88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 μg/mL, 518.4 ± 115.8 μg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 μg/mL, 611.0 ± 42.5 μg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and β-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05).

CONCLUSIONSPancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.

Acinar Cells ; drug effects ; metabolism ; Animals ; Blotting, Western ; Calcium ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fluorescence ; Gene Expression Regulation ; drug effects ; Inositol 1,4,5-Trisphosphate ; metabolism ; Pancreas ; pathology ; Pancreatitis, Acute Necrotizing ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Receptors, Cholecystokinin ; genetics ; metabolism ; Signal Transduction ; drug effects ; Type C Phospholipases ; metabolism

The gingival epithelium of the oral cavity is constantly exposed to exogenous stimuli such as bacterial toxins, allergens, and thermal changes. These exogenous stimuli are resisted by innate host defense in gingival epithelial cells. However, it is unclear exactly how the exogenous stimuli affect detrimentally on the human gingival epithelial cells. Here, we investigated whether the allergen, such as house dust mite (HDM) extract, is linked to Ca2+ signaling and proinflammatory cytokine expression in primary cultured human gingival epithelial cells. HDM extract induced an increase in intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner. Extracellular Ca2+ depletion did not affected on the HDM extract-induced increase in [Ca2+]i. The HDM extract-induced increase in [Ca2+]i was abolished by the treatment with U73122 and 2-APB, which are inhibitors of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptor. Moreover, HDM extract induced the mRNA expression of pro-inflammatory cytokine, interleukin (IL)-8. These results suggest that HDM extract triggers PLC/IP3-dependent Ca2+ signaling and IL-8 mRNA expression in primary cultured human gingival epithelial cells.
Allergens ; Bacterial Toxins ; Epithelial Cells* ; Epithelium ; Humans ; Inositol 1,4,5-Trisphosphate ; Interleukin-8* ; Interleukins ; Mouth ; Pyroglyphidae* ; RNA, Messenger ; Type C Phospholipases