Involvement Of Inositol Research Articles

Oxidative stress impairs the calcification ability of human dental pulp cells

BackgroundThe relationship between internal root resorption and oxidative stress has not yet been reported. This study aimed to add molecular insight into internal root resorption. The present study was conducted to investigate the effect of hydrogen peroxide (H2O2) as an inducer of oxidative stress on the calcification ability of human dental pulp cells (hDPCs) and the involvement of inositol 1, 4, 5-trisphosphate (IP3).Material and methodshDPCs (Lonza, Basel, Switzerland) were exposed to H2O2. Cell viability and reactive oxygen species (ROS) production were then evaluated. To investigate the effect of H2O2 on the calcification ability of hDPCs, real-time PCR for alkaline phosphatase (ALP) mRNA expression, ALP staining, and Alizarin red staining were performed. Data were compared with those of hDPCs pretreated with 2-aminoethyldiphenylborate (2-APB), which is an IP3 receptor inhibitor.ResultsH2O2 at concentrations above 250 µM significantly reduced cell viability (P < 0.01). More ROS production occurred in 100 µM H2O2-treated hDPCs than in control cells (P < 0.01). 2-APB significantly decreased the production (P < 0.05). H2O2-treated hDPCs showed significant reductions in ALP mRNA expression (P < 0.01), ALP activity (P < 0.01), and mineralized nodule deposition compared with negative control cells (P < 0.01). 2-APB significantly inhibited these reductions (P < 0.01, P < 0.05 and P < 0.01, respectively). Data are representative of three independent experiments with three replicates for each treatment and values are expressed as means ± SD.ConclusionTo the best of our knowledge, this is the first study documenting the involvement of IP3 signaling in the calcification ability of human dental pulp cells impaired by H2O2.

Involvement of Inositol 1,4,5-Trisphosphate Formation in the Voltage-Dependent Regulation of the Ca2+Concentration in Porcine Coronary Arterial Smooth Muscle Cells

The involvement of inositol 1,4,5-trisphosphate (IP(3)) formation in the voltage-dependent regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) was examined in smooth muscle cells of the porcine coronary artery. Slow ramp depolarization from -90 to 0 mV induced progressive [Ca(2+)](i) increase. The slope was reduced or increased in the presence of Cd(2+) or (±)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]-phenyl)pyridine-3-carboxlic acid methyl ester (Bay K 8644), respectively. The decrease in [Ca(2+)](i) via the membrane hyperpolarization induced by K(+) channel openers (levcromakalim and Evans blue) under current clamp was identical to that under voltage clamp. The step hyperpolarization from -40 to -80 mV reduced [Ca(2+)](i) uniformly over the whole-cell area with a time constant of ∼10 s. The [Ca(2+)](i) at either potential was unaffected by heparin, an inhibitor of IP(3) receptors. Alternatively, [Ca(2+)](i) rapidly increased in the peripheral regions by depolarization from -80 to 0 mV and stayed at that level (∼400 nM) during a 60-s pulse. When the pipette solution contained IP(3) pathway blockers [heparin, 2-aminoethoxydiphenylborate, xestospongin C, or 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U73122)], the peak [Ca(2+)](i) was unchanged, but the sustained [Ca(2+)](i) was gradually reduced by ∼250 nM within 30 s. In the presence of Cd(2+), a long depolarization period slightly increased the [Ca(2+)](i), which was lower than that in the presence of heparin alone. In coronary arterial myocytes, the sustained increase in the [Ca(2+)](i) during depolarization was partly caused by the Ca(2+) release mediated by the enhanced formation of IP(3). The initial [Ca(2+)](i) elevation triggered by the Ca(2+) influx though voltage-dependent Ca(2+) channels may be predominantly responsible for the activation of phospholipase C for IP(3) formation.

First molecular evidence that inositol trisphosphate signaling contributes to infarct size reduction with preconditioning

Considerable attention has focused on the role of protein kinase C (PKC) in triggering the profound infarct-sparing effect of ischemic preconditioning (PC). In contrast, the involvement of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)], the second messenger generated in parallel with the diacylglycerol-PKC pathway, remains poorly understood. We hypothesized that, if Ins(1,4,5)P(3) signaling [i.e., release of Ins(1,4,5)P(3) and subsequent binding to Ins(1,4,5)P(3) receptors] contributes to PC-induced cardioprotection, then the reduction of infarct size achieved with PC would be attenuated in mice that are deficient in Ins(1,4,5)P(3) receptor protein. To test this concept, hearts were harvested from 1) B6C3Fe-a/a-Itpr-1(opt+/-)/J mutants displaying reduced expression of Ins(1,4,5)P(3) receptor-1 protein, 2) Itpr-1(opt+/+) wild types from the colony, and 3) C57BL/6J mice. All hearts were buffer-perfused and randomized to receive two 5-min episodes of PC ischemia, pretreatment with d-myo-Ins(1,4,5)P(3) [sodium salt of native Ins(1,4,5)P(3)], the mitochondrial ATP-sensitive K(+) channel opener diazoxide, or no intervention (controls). After the treatment phase, all hearts underwent 30-min global ischemia followed by 2 h of reperfusion, and infarct size was delineated by tetrazolium staining. In both wild-type and C57BL/6J cohorts, area of necrosis in hearts that received PC, d-myo-Ins(1,4,5)P(3), and diazoxide averaged 28-35% of the total left ventricle (LV), significantly smaller than the values of 52-53% seen in controls (P < 0.05). In contrast, in Itpr-1(opt+/-) mutants, protection was only seen with diazoxide: neither PC nor d-myo-Ins(1,4,5)P(3) limited infarct size (52-58% vs. 56% of the LV in mutant controls). These data provide novel evidence that Ins(1,4,5)P(3) signaling contributes to infarct size reduction with PC.

Involvement of Inositol 1,4,5-Trisphosphate in Nicotinic Calcium Responses in Dystrophic Myotubes Assessed by Near-plasma Membrane Calcium Measurement

In skeletal muscle cells, plasma membrane depolarization causes a rapid calcium release from the sarcoplasmic reticulum through ryanodine receptors triggering contraction. In Duchenne muscular dystrophy (DMD), a lethal disease that is caused by the lack of the cytoskeletal protein dystrophin, the cytosolic calcium concentration is known to be increased, and this increase may lead to cell necrosis. Here, we used myotubes derived from control and mdx mice, the murine model of DMD, to study the calcium responses induced by nicotinic acetylcholine receptor stimulation. The photoprotein aequorin was expressed in the cytosol or targeted to the plasma membrane as a fusion protein with the synaptosome-associated protein SNAP-25, thus allowing calcium measurements in a restricted area localized just below the plasma membrane. The carbachol-induced calcium responses were 4.5 times bigger in dystrophic myotubes than in control myotubes. Moreover, in dystrophic myotubes the carbachol-mediated calcium responses measured in the subsarcolemmal area were at least 10 times bigger than in the bulk cytosol. The initial calcium responses were due to calcium influx into the cells followed by a fast refilling/release phase from the sarcoplasmic reticulum. In addition and unexpectedly, the inositol 1,4,5-trisphosphate receptor pathway was involved in these calcium signals only in the dystrophic myotubes. This surprising involvement of this calcium release channel in the excitation-contraction coupling could open new ways for understanding exercise-induced calcium increases and downstream muscle degeneration in mdx mice and, therefore, in DMD.

Inositol is specifically involved in the sexual program of the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is a natural auxotroph for inositol and fails to grow in the complete absence of it. It was previously reported that a small concentration of inositol in the culture medium supports vegetative growth, but not mating and sporulation, and a tenfold of that concentration also supports mating and sporulation. The purpose of the present work was to investigate whether a moderate inositol starvation specifically affected events of the sexual program of development. A homothallic culture grown to the stationary phase in medium with a small inositol concentration was sterile but cells in the stationary phase of growth synchronously entered and completed the sexual cycle when inositol was added, without need of previous cell divisions. This suggests the involvement of inositol in a mechanism (or mechanisms) of the sexual program. The events of the program that were affected by inositol starvation were investigated. Commitment to mating and production of pheromone M were shown not to be inositol-dependent. A diploid strain homozygous at the mating-type locus and carrying a pat1-114 temperature-sensitive mutation in homozygous configuration sporulated under inositol starvation at the restrictive temperature; therefore starvation did not directly affect meiosis or sporulation. In contrast, production of pheromone P and the response of cells to pheromones were found to be inositol-dependent. The possibility that inositol or one of its derivative compounds is involved in pheromone P secretion and in pheromone signal reception is discussed.

Involvement of inositol 1,4,5-trisphosphate-regulated stores of intracellular calcium in calcium dysregulation and neuron cell death caused by HIV-1 protein tat.

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.

Ca2+-activated K+ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia.

Ca2+-activated K+ currents in rat locus coeruleus neurons induced by experimental ischemia, anoxia, and hypoglycemia. J. Neurophysiol. 78: 2674-2681, 1997. The effects of metabolic inhibition on membrane currents and N-methyl--aspartic acid (NMDA)-induced currents were investigated in dissociated rat locus coeruleus (LC) neurons by using the nystatin perforated patch recording mode under voltage-clamp conditions. Changes in the intracellular Ca2+ concentration ([Ca2+]i) during the metabolic inhibition were also investigated by using the microfluometry with a fluorescent probe, Indo-1. Removal of both the oxygen and glucose (experimental ischemia), deprivation of glucose (hypoglycemia), and a blockade of electron transport by sodium cyanide (NaCN) or a reduction of the mitochondrial membrane potential with carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone(FCCP) as experimental anoxia all induced a slowly developing outward current (IOUT) at a holding potential of -40 mV. The application of 10(-4) M NMDA induced a rapid transient peak and a successive steady state inward current and a transient outward current immediately after washout. All treatments related to metabolic inhibition increased the NMDA-induced outward current(INMDA-OUT) and prolonged the one-half recovery time of INMDA-OUT. The reversal potentials of both IOUT and INMDA-OUT were close to the K+ equilibrium potential (EK) of -82 mV. Either charybdotoxin or tolbutamide inhibited the IOUT and INMDA-OUT, suggesting the contribution of Ca2+-activated and ATP-sensitive K+ channels, even though the inhibitory effect of tolbutamide gradually diminished with time. Under the metabolic inhibition, the basal level of [Ca2+]i was increased and the one-half recovery time of the NMDA-induced increase in [Ca2+]i was prolonged. The IOUT induced by NaCN was inhibited by a continuous treatment of thapsigargin but not by ryanodine, indicating the involvement of inositol 1,4, 5-trisphosphate (IP3)-induced Ca2+ release (IICR) store. These findings suggest that energy deficiency causes Ca2+ release from the IICR store and activates continuous Ca2+-activated K+ channels and transient ATP-sensitive K+ channels in acutely dissociated rat LC neurons.

Involvement of inositol 1,4,5-triphosphate and protein kinase C in thrombin-induced contraction of porcine pulmonary artery

The role of the intracellular messengers inositol 1,4,5-triphosphate (IP 3) and protein kinase C (PKC) in the thrombin (3 U/mL)-induced contraction of endothelium-denuded porcine pulmonary arteries was investigated. Thrombin induced a sustained contractile response with an initial transient increase in IP 3 to about 160% of the unstimulated control. Omission of extracellular Ca 2+ or preincubation with verapamil (10 μmol/L) reduced the maximum of contraction without significantly affecting the thrombin-induced increase in IP 3. To evaluate the role of PKC for the contractile response, the PKC was activated directly by phorbol 12,13-dibutyrate (PDBu, 50 nmol/L). The phorbol ester produced a slowly increasing tonic contraction without any changes in the basal IP 3 level. There was a moderate inhibition of PDBu-induced contractions in Ca 2+-free solution, while they were not inhibited after preincubation with verapamil. Preincubation with the PKC inhibitor staurosporine (50 nmol/L) significantly reduced the PDBu-induced contraction (by about 80%). In thrombin-stimulated vessels staurosporine only inhibited the tonic phase of the contractile response whereas the increase in IP 3 and the phasic component of contraction were still evident. These results suggest that IP 3 and PKC are involved in the thrombin-induced contraction. The phasic component of contraction is associated with the generation of IP 3; the tonic component might be due to the activation of PKC.

Multistep mechanism of polarized Ca2+ wave patterns in hepatocytes.

The spatial organization of cytosolic Ca2+ (Ca2+i) signals is thought to be important for regulation of cell function. In epithelial cells, the involvement of inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release in evoking Ca2+i signals is appreciated, but the location of IP3-sensitive Ca2+ stores and the role of Ca(2+)-induced Ca2+ release (CICR) for Ca2+ signaling are less defined. Here, we demonstrate that IP3 receptors are localized to the apical region in hepatocytes. We also show that hormone-induced Ca2+i waves propagate across the cell at a rate that depends on mobilization of Ca2+ stores that are sensitive to caffeine, ryanodine, and dantrolene, and that these agents, at concentrations that inhibit CICR, decrease the magnitude of Ca2+i signals. Furthermore, Ca2+i wave speed is not reduced in Ca(2+)-free medium. These findings suggest that Ca2+i signals in epithelial cells begin as apical-to-basal Ca2+i waves that result from sequential release of Ca2+, first from IP3-sensitive stores in the apex and then from Ca(2+)-sensitive stores distributed across the remainder of the cell.

Involvement of inositol 1,4,5-trisphosphate-mediated Ca2+release in early and late events of mouse egg activation

Sperm-induced activation of mammalian eggs is associated with a transient increase in the concentration of intracellular Ca2+. The role of inositol 1,4,5-trisphosphate (IP3)-mediated release of Ca2+ from intracellular stores during mouse egg activation was examined in the present study by determining the effects of microinjected monoclonal antibody (mAb) 18A10, which binds to the IP3 receptor and inhibits IP3-induced Ca2+ release, on endpoints of egg activation following insemination. The antibody inhibited in a concentration-dependent manner the ZP2 to ZP2f conversion that is involved in the zona pellucida block to polyspermy, as well as the ZP2 to ZP2f conversion promoted by microinjected IP3 in non-inseminated eggs. As anticipated, inseminated eggs that had been microinjected with the antibody were polyspermic. In addition, the antibody inhibited the fertilization-associated decrease in H1 kinase activity and pronucleus formation, and the concentration dependence for inhibition of these events was similar to that observed for inhibiting the ZP2 to ZP2f conversion. Last, the antibody inhibited the fertilization-induced recruitment of maternal mRNAs and post-translational modifications of proteins. In each case, eggs microinjected with the mAb 4C11, which also binds to the IP3 receptor but does not inhibit IP3-induced Ca2+ release, had no inhibitory effect on fertilization and egg activation. Results of these studies suggest that IP3-mediated Ca2+ release is essential for both early and late events of mouse egg activation.

Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells.

ADP and ATP, in the 1-100 microM range of concentrations, increased the formation of inositol phosphates in bovine aortic endothelial cells. The accumulation of inositol trisphosphate in response to adenine nucleotides was rapid (maximum at 15 s) and transient. This material was identified as the biologically active isomer inositol 1,4,5-trisphosphate on the basis of its retention time by high-performance liquid chromatography on an anion-exchange resin. AMP and adenosine have no effect on inositol phosphates. The action of ATP and ADP was mimicked with an equal potency and activity by their phosphorothioate analogs, ATP gamma S and ADP beta S, and with a lower potency by adenosine 5'-(beta,gamma-imido)triphosphate, whereas adenosine 5'-(alpha,beta-methylene)triphosphate, was inactive. In the same range of concentrations, ADP and ATP induced an efflux of 45Ca2+ from prelabeled bovine aortic endothelial cells and increased the fluorescence emission by cells loaded with quin-2. Here, too, AMP and adenosine were completely inactive. The outflow of 45Ca2+ induced by ADP was partially maintained in a calcium-free medium. These data suggest that in aortic endothelial cells, P2-purinergic receptors, of the P2Y subtype, are coupled to the hydrolysis of phosphatidylinositol bisphosphate by a phospholipase C. It is likely that the release of prostacyclin and endothelium-derived relaxing factor in response to ADP and ATP is a consequence of this initial event.

The involvement of inositol 1,4,5-trisphosphate and calcium in the two-component response to acetylcholine in Xenopus oocytes.

1. The membrane response to acetylcholine (ACh), inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ was studied in Xenopus laevis oocytes under voltage-clamp conditions. 2. Shallow, submembranal injections of IP3 in the animal hemisphere of the oocyte evoked a two-component response comprised of a rapid, transient component followed by a slow, sustained component. 3. When the injection pipette was inserted further into the cell (to 300 microns below the cell membrane), the fast component diminished and the slow component remained unchanged or even increased. 4. The rapid component exhibited an apparent higher sensitivity to IP3 compared to the slow component. 5. The two components of the IP3 response were retained in a Ca2+-free environment. 6. Injection of a single large dose (20-50 pmol) of CaCl2 into the oocyte evoked a typical two-component response, whereas repetitive threshold doses (0.1 pmol CaCl2) elicited large current fluctuations which developed into a small depolarization current. 7. The delay in the peak of the slow component of the response to either IP3 or to CaCl2 injections appeared too long to be accounted for by diffusion alone. 8. Depletion of oocyte Ca2+ by the divalent cation ionophore A23187 (greater than 1 microM) inhibited the response to ACh and IP3. Low concentrations of A23187 selectively inhibited the rapid component of the ACh response, though not the rapid component of the IP3 response. 9. Our data suggest that the two-component membrane response to ACh in Xenopus oocytes can be accounted for by ACh-induced elevation of IP3 and subsequent IP3-induced release of intracellular Ca2+.