Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa / 亚洲男科学杂志(英文版)

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.

Calcium Signaling in Salivary Secretion

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.

Effect of isoflurane on apoptosis of SH-SY5Y cells transfected with APPsw gene and the role of inositol 1,4,5-triphosphate receptors / 中华麻醉学杂志

Objective To evaluate the effect of isoflurane on the apoptosis of SH-SYSY cells transfected with APPsw gene and the role of inositol 1,4,5-triphosphate (IP3) recepters.Methods The SH-SYSY ceils transfected with APPsw gene were seeded in culture flasks with the density of 1.2 × 104/cm2.The cells were randomly divided into 4 groups (n =6 each):control group (group C),IP3 receptor antagonist group (group Ⅹ),isoflurane group (group Ⅰ) and isoflurane + IP3 receptor antagonist group (group Ⅰ + Ⅹ).After the cells were cultured for 24 h and attached to the wall,the cells were cultured routinely in group C,and Xestospongin C 100 nmol/L (IP3 receptor antagonist) was added to DMEM culture medium in groups X and Ⅰ + X,and 30 min later the cells were exposed to 1.2 ％ sevoflurane for 8 h in groups Ⅰ and Ⅰ + X.The cells were collected for examination of the ultrastructure and for determination of cell apoptosis,intracellular free calcium ion concentration [Ca2 +] i (by flow cytometry) and expression of IP3 receptor protein (by Western blot).The apoptosis rate was calculated.Results Compared with group C,there was no significant change in the apoptosis rate,[Ca2 +]i or IP3 receptor protein expression in group Ⅹ (P ＞ 0.05),while the cell apoptosis rate and [Ca2 +] i were significantly increased and IP3 receptor protein expression was up-regulated in groups I and Ⅰ + Ⅹ (P ＜ 0.05 or 0.01).Compared with group Ⅰ,cell apoptosis rate and [Ca2+]i were significantly decreased and IP3 receptor protein expression was down-regulated in group Ⅰ + Ⅹ (P ＜ 0.01).The pathological changes of the cells happened in groups Ⅰ and Ⅰ + Ⅹ,and the pathological changes were severer in group Ⅰ than in group Ⅰ + Ⅹ.Conclusion Isoflurane can induce apoptosis of SH-SY5Y cells transfected with APPsw gene through increasing [Ca2+]i and up-regulating IP3 receptor protein expression.

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

The receptor activator of NF-kappaB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-kappaB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) delta, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

Microsomal Ca2+ flux modulation as an indicator of heavy metal toxicity.

Inositol 1,4,5-trisphosphatee (IP3), an intracellular messenger, releases Ca2+ from microsomes. Ca2+ plays a major role in regulating various cellular events like neural transmission and regulation of hormones and growth factors. Aluminum (Al), lead (Pb) and mercury (Hg) were reported to alter Ca2+-regulated events thereby causing neurotoxicity. Hence, an attempt was made characterize IP3 mediated Ca2+ release from rat brain microsomes under the influence of Al, Pb and Hg. Different concentrations of metals were tested over a designated time scale and their effects on IP3 mediated Ca2+ release from microsomes were monitored using Fura-2 technique. All the three metals inhibited IP3 mediated Ca2+ release, Pb being more potent. The order of potency of these three metals was Pb>Hg>Al. Except for Al, both Hg and Pb independently released Ca2+ from microsomes. Re-uptake of Ca2+ into microsomes was inhibited by all the three metals, Pb being more potent. Microsomal Ca2+-ATPase activity was also inhibited by all the three metals. These results suggest that neurotoxicity exerted by Al, Pb and Hg may be due to the interference of these metals with IP3 mediated calcium release and also interfering with the microsomal Ca2+ sequestration mechanism. Differential effects of heavy metal induced changes in Ca2+ flux can be used as an index of relative toxicity.

Effects of Hirudin on Ventricular Arrhythmia after Acute Myocardial Infarction in Rats / 中山大学学报(医学科学版)

[Objective] To determine the effects and possible mechanism of the thrombin antagonist r-RGD-Hirudin (HIR) on ventricular arrhythmia(VA) after acute myocardial infarction (AMI). [Methods] Seventy adult male Sprague-Dawley rats were randomly subjected to the 10 groups according to duration of left coronary occlusion: HIR 0 min, HIR 5 rain, HIR 10 min, HIR 20 min, HIR 30 min, and normal saline(NS) 0 min, NS 5 min, NS 10 min, NS 20 min, NS 30 min; and the average of every group is 7 rats. Acute myocardial infarction was produced by the occlusion of the left anterior descending coronary artery, then the measurements of arrhythmia and infarction sizing by Evans blue were assessed as well as the expression of three isoforms of inositol 1,4,5-trisphosphate receptors (IP3Rs) mRNA in isehemic myocardium by reverse transeriptase polymerase chain reactions (RT-PCR). [Results] Compared with NS groups, the measurements of VA in HIR were reduced significantly in 5 to 20 minutes after AMI (P<0.05). The incidence of VA was all positive related to the expression of three isoforms of IP3Rs mRNA (P<0.01). Compared with NS groups, the expression of type2,inositol 1,4,5-trisphosphate receptor (IP3R2) mRNA at 10 min and type3, inositol 1,4,5-trisphosphate receptor mRNA (IP3R3) at 10 min and 20 min after AMI were significant decreased (P<0.05) in HIR groups. [Conclusion] The thrombin antagonist r-RGD-Hirudin exerts its myocardial protection against ventricular arrhythmia after acute myocardial infarction possible through IP3R2 and IP3R3 and not typel, inositol 1,4,5-trisphosphate receptor (IP3R1).

Apoptotic effects of inositol hexaphosphate on biomarker Itpr3 in induced colon rat carcinogenesis

PURPOSE: To study the effect of the modulation of inositol hexaphosphate (IP6) in the biological immunohistochemistry expression of cellular signaling marker apoptosis, in model of carcinogenesis of colon induced by azoxymethane (AOM). METHODS: Wistar rats (N=112) distributed in 4 groups (n=28): Control; B, AOM (5 mg kg-1, 2x, to break week 3); C, IP6 (in water 1 percent, six weeks); D, IP6+AOM. Weekly euthanasia (n=7), from week three. Immunohistochemistry of ascendant colon with biological marker inositol 1,4,5 triphosphate receptor type III (Itpr3). Quantification of the immune-expression with use of computer-assisted image processing. Analysis statistics of the means between groups, weeks in groups, groups in weeks, and established significance when p<0.05. RESULTS: One proved significant difference between groups in the expression of Itpr3, p<0.0001; with Itpr3 reduction of BxD group, p<0.001. CONCLUSION: Inositol hexaphosphate promotes modulation of biological markers with reduction of Itpr3 in carcinogenesis of colon.

OBJETIVO: Estudar os efeitos da modulação do inositol hexafosfato (IP6) na expressão imunoistoquímica de marcador biológico de sinalização celular de apoptose, em modelo de carcinogênese induzida pelo azoximetano (AOM). MÉTODOS: Ratos Wistar (N=112) distribuídos em 4 grupos (n=28): A, controle; B, AOM (5 mg Kg-1, 2x, a partir semana 3); C, IP6 (em água a 1 por cento, seis semanas); D, IP6+AOM. Eutanásia semanal (n=7), a partir de semana três. Imunoistoquímica de colo ascendente com marcador biológico inositol 1,4,5 trisphosphate receptor type III (Itpr3). Quantificação da imunoexpressão com uso de processamento imagem assistida computador. Análise estatística da expressão média entre grupos, semanas em grupos e grupos em semanas, e estabelecido significância quando p<0.05. RESULTADOS: Evidenciou-se diferença significante entre grupos na expressão de Itpr3, p<0.0001; com diminuição Itpr3 de grupo BxD, p<0.001. CONCLUSÃO: O inositol hexafostato promove a modulação de marcador biológico com diminuição Itpr3 em carcinogênese de colo.

Change of cardiac myocyte nuclear inositol 1,4,5- trisphospate receptor binding proterties in rat with myocardium ischemic reperfusion / 中国药理学通报

Aim Observing the alteration of cardiac myocyte nuclear inositol 1,4,5-trisphosphate receptor (IP_3R)binding proterties in rat subjected to myocardium ischemic reperfusion is to make it clear whether this change is involved in the molecule mechanism of cell apoptosis of rat with myocardial ischemic reperfusion. Method Apoptosis index of myocardial cell was determined using TUNEL assay.Extracting of cardiac myocyte nucleus was accomplished by saccharose density gradient centrifugation method,the binding proterties of nuclear IP_3R in different conditions were detected by radioligand binding assay.Results ①Myocardial cell apoptosis index in rat heart underwent 30 min regional ischemia and 3 h reperfusion was distinctly increased compared with sham-operated group(P

Construction of lentivirus RNAi vector pLenti6/V5-DEST-IP_3R1 and identification of its silencing effects / 解放军医学杂志

Objective To construct lentiviral expression vector of rat IP3R1 gene,and identify its silencing effect by using PC12 cell lines. Methods Oligo DNA sequences of 4 pairs of miRNA,named as miRNA1,miRNA2,miRNA3 and miRNA4,were designed according to IP3R1 gene sequence (GenBank:NM_001007235). The single strand of oligo DNA was annealed to form double strand DNA,and then connected with the empty plasmid pcDNA TM 6.2-GW/EmGFP-miR. By using gateway technology,the expression vector pcDNA TM 6.2-GW/EmGFP-miR-IP3R1 was linked into lentiviral destination vector pLenti6/V5-DEST to form the lentiviral expression vector pLenti6/V5-DEST-IP3R1,then it was transformed into infectious lentiviral particles and to infect PC12 cell lines. Silencing effect of gene IP3R1 was detected by Real-time PCR and Western blot. Results The sequence of expression vector pcDNA TM 6.2-GW/EmGFP-miR-IP3R1 was proved correct using sequencing method. After the transfection of letivirus vector pLenti6/V5-DEST-IP3R1 into PC12 cell lines,the IP3R1 mRNA and protein level were down-regulated 48h later,of which miRNA2 and miRNA3 sequence showed the best silencing effect,and the expression of IP3R1 in the blank control and negative control showed no significant changes. Conclusions Lentiviral expression vector pLenti6/V5-DEST-IP3R1 was constructed successfully. pLenti6/V5-DEST-IP3R1 may render the IP3R1 expression in PC12 cell lines down-regulated,and it provides a foundation for studying the function of calcium release channel IP3R1.

The effect of kainic acid induced convulsions on the expression of inositol 1,4,5-trisphosphate receptor mRNA in the rat brain / 대한해부학회지

In this study, the effect of systemic administration of kainic acid (KA) on the expression of inositol 1,4,5-trisphosphate receptor mRNA in the rat brain was investigated with in situ hybridization histochemistry. After the injection of KA in a convulsive dose (10 mg/kg i.p.), inositol 1,4,5-trisphosphate receptor mRNA was reduced significantly in dentate gyrus, cerebral cortex, and caudate-putamen and moderately in CA1-CA3 areas of hippocampus and cerebellum. In dentate gyrus, the expression of inositol 1,4,5-trisphosphate receptor mRNA was significantly decreased at 6 h, lowest level at 9 h, after that the expression was gradually recovered and returned to basal level at 72 h after KA injection. However, in the CA1-CA3 areas of the hippocampus, cerebral cortex, and caudate-putamen, the expression of inositol 1,4,5-trisphosphate receptor mRNA was abruptly decreased at 9 h and almost return to basal level at 24 h after KA injection. The significant repression of inositol 1,4,5-trisphosphate receptor mRNA in cerebellum was only found at 9 h after KA injection. But significant change of inositol 1,4,5-trisphosphate receptor mRNA was not found in the brains of rats treated with NMDA receptor blocker, MK-801, followed by KA injection. These observations suggest that the inositol 1,4,5-trisphosphate receptor is one of the genes whose expression can be altered by KA treatment and the NMDA receptor is related with this alternation.

Mechanism of Inhibition of propofol on intracellular calcium increase in aortic smooth muscle cells / 中华麻醉学杂志

Objective:To test the effects of propofol on intracellular calcium free concentration ([Ca~(2+)]i) and inositol 1,4,5-triphosphate (IP_3) biological synthesis induced by norepinephrine (NE) and 5-hydroxytryptamine (5-HT) in aortic smooth muscle cells (ASMC)of rats for the mechanism of relaxtion of propofol on vascular smooth muscle.Method: Using the flurospectrophotometry and Fura-2/AM loading method,the changes of [Ca~(2+)]i levels in primary culture ASMC were measured,and using the specific, IP_3 assay system and isotope radioactive protein binding experiment IP_3 production levels in aortic smooth muscle were measured. Result:The baselines of [Ca~(2+)]i was decreased when primary culture ASMC was pretreated with propofol in 72 hours. Propofol inhibited [Ca~(2+)]i increase induced by NE and 5-HT in dose-dependent way. With extracellular calcium free or calcium channel blocker(Verapamil),inhibition of propofol on NE and 5-HT increasing [Ca~(2+)]i levels were decreased,but could not be cancelled. Propofol depressed IP_3 biological synthesis induced by NE and 5-HT in dose-dependent way. Conclusion:Relaxation of propofol on aortic smooth muscle is closely related to inhibiting IP_3- induced calcium release to decrease intracellular calcium concentration.

Inhibitory effect of ATP on proliferative signaling in immortalized human fibroblasts / 中国病理生理杂志

AIM: To investigate the inhibitory effects of ATP on proliferation signaling in immortalized human fibroblasts. METHODS: Immortalized human fibroblasts were treated with ATP, ATP conbined with calcium or potassium channel antagonists, respectively. The intracelluar-free calcium ([Ca 2+ ]i), inositol 1,4,5-trisphosphate(IP 3) levels and cell viability were detected at different time points. RESULTS: ATP significantly increased the [Ca 2+ ]i and decreased the IP 3 level in immortalized human fibroblasts, especially at initial stage ( P

Role of IP_3 in the signal-transduction of IL-2—induced proliferation of human T Iymphocytes / 中国免疫学杂志

The role that inositol-1, 1.5 -trisphosphate (IP, ) (?)lays in the proliferation of T lymphocyte induced by IL- 2 was studied. In resting T lymphocytes, IL- 2 did notalter intracellular concentration of IP_3, but Con A stimulation increased IP_3 by 45%. In human IL-2 sensitive T cells, which expressed IL-2 receptors by 83%, the Changes in intracellular IP_3 was dependent upon IL-2 concentrations. The IP_3 increased at IL-2 concentrations of 10 and 50 U/ml and the maximal response of 60% was attained at the concentration of 50 U/ml. At IL-2 concentration of 100 U/ml no increase in IP_3 was observed. After binding of anti-Tac McAb to IL-2 receptors of T lymphocyte the increase in IP_3 at IL-2 concentrations of 10 and 50 U/ml was significantly attenuated. It has been suggested that IP_3 could play the important role in signa l-transduction of IL-2-induced proliferation of T Lymphocyte, which is related to the IL-2 concentrations in T cells culture and the expression of IL-2 receptors on T lymphocyte.