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PubMed2001

Role of Ins(1,4,5)P3, cADP-ribose and nicotinic acid-adenine dinucleotide phosphate in Ca2+ signalling in mouse submandibular acinar cells.

cADP-ribose (cADPr) and nicotinic acid-adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca(2+) mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P(3) in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca(2+)-dependent K(+) and Cl(-) currents. Both Ins(1,4,5)P(3) and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10-20 nM ACh) or second messenger [1-10 microM Ins(1,4,5)P(3) or cADPr] elicited a sporadic transient activation of the Ca(2+)-dependent currents; mid-range concentrations [50-500 nM ACh, 50 microM Ins(1,4,5)P(3) or 50-100 microM cADPr] elicited high-frequency (approx. 2 Hz) trains of current spikes; and high concentrations [more than 500 nM ACh, more than 50 microM Ins(1,4,5)P(3) or more than 100 microM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P(3)R antagonist (heparin). NAADP (50 nM to 100 microM) did not itself activate the Ca(2+)-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P(3)R and RyR are involved in the propagation of the Ca(2+) signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

Acetylcholine↗
PubMed2001

Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation.

The pleckstrin homology domain of phospholipase Cdelta1 (PH(PLCdelta)) binds Ins(1,4,5)P(3) and PtdIns(4,5)P(2) specifically, and can be used to detect changes in Ins(1,4,5)P(3) in single cells. A fusion construct of PH(PLCdelta) and enhanced green fluorescent protein (EGFP-PH(PLCdelta)) associates with the plasma membrane due to its association with PtdIns(4,5)P(2). However, PH(PLCdelta) has greater affinity for Ins(1,4,5)P(3) than PtdIns(4,5)P(2), and translocates to the cytosol as Ins(1,4,5)P(3) levels rise. Prolonged activation of group I metabotropic glutamate receptor 1alpha expressed in Chinese-hamster ovary cells or endogenous M(3) muscarinic receptors in SH-SY5Y neuroblastoma cells gave an initial transient peak in translocation, followed by a sustained plateau phase. This closely followed changes in cell population Ins(1,4,5)P(3) mass, but not PtdIns(4,5)P(2) levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P(3). Graded changes in Ins(1,4,5)P(3) in Chinese-hamster ovary-lac-mGlu1alpha cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP-PH(PLCdelta) showed that changes in intracellular Ca(2+) concentration closely paralleled Ins(1,4,5)P(3) production. Moreover, Ins(1,4,5)P(3) accumulation and intracellular Ca(2+) mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.

Animals↗
PubMed2005

The Ins(1,3,4)P3 5/6-kinase/Ins(3,4,5,6)P4 1-kinase is not a protein kinase.

Among inositol phosphate kinases, Ins(3,4,5,6)P4 1-kinase has been considered to be an outsider with disparate sequence, a proclaimed capacity to also phosphorylate proteins and apparent 1-phosphatase activity. Such multifunctionality, coupled with ignorance of its operational domains, complicates any mechanistic rationale behind literature reports that Ins(3,4,5,6)P4 1-kinase regulates apoptosis, salt and fluid secretion, and transcription. We have expressed poly(His)-tagged human Ins(3,4,5,6)P4 1-kinase in Sf9 insect cells and purified the enzyme using Ni-agarose chromatography. Protein kinase activity was eluted from the Ni-agarose column, but this did not co-elute with the Ins(3,4,5,6)P4 1-kinase, indicating that the protein kinase and inositol kinase activities belong to separate proteins. To pursue this conclusion, we prepared catalytically inactive mutants of the Ins(3,4,5,6)P4 1-kinase by identifying and targeting the ATP-binding site. Our strategy was based on sequence alignments suggesting homology of the Ins(3,4,5,6)P4 1-kinase with ATP-grasp metabolic enzymes. Individual mutation of four candidate MgATP-binding participants, Lys157, Asp281, Asp295 and Asn297, severely compromised Ins(3,4,5,6)P4 1-kinase activity. Yet, these mutations did not affect the protein kinase activity. We conclude that the Ins(3,4,5,6)P4 1-kinase is not a protein kinase, contrary to earlier reports [e.g. Wilson, Sun, Cao and Majerus (2001) J. Biol. Chem. 276, 40998-41004]. Elimination of protein kinase activity from the enzyme's repertoire and recognition of its ATP-grasp homology together indicate that structural, functional and catalytic relationships between Ins(3,4,5,6)P4 1-kinase and other inositol phosphate kinases are closer than previously thought [Gonzalez, Schell, Letcher, Veprintsev, Irvine and Williams (2004) Mol. Cell 15, 689-701].

Adenosine Triphosphate↗
PubMed1992

Ins(1,3,4,5)P4 promotes sustained activation of the Ca(2+(-dependent Cl- current in isolated mouse lacrimal cells.

Infusion of 50 microM-Ins(1,3,4,5)P4 in addition to 500 microM-Ins(1,4,5)P3 into mouse lacrimal cells via a patch-clamp pipette promoted sustained activation of the Ca(2+)-dependent Cl- current, which could not be achieved with 500 microM-Ins(1,4,5)P3 alone. It has been proposed that Ins(1,3,4,5)P4 facilitates Ca2+ influx in the presence of Ins(1,4,5)P3 [Morris, Gallacher, Irvine & Petersen (1987) Nature (London) 330, 653-655], but a subsequent study in mouse lacrimal cells [Bird, Rossier, Hughes, Shears, Armstrong & Putney (1991) Nature (London) 352, 162-165] showed that a high concentration of Ins(1,4,5)P3 could mobilize both intra- and extra-cellular Ca2+ in the absence of Ins(1,3,4,5)P4. My data confirm these findings, but also show that Ins(1,3,4,5)P4 can stimulate additional Ca2+ influx even when the Ins(1,4,5)P3-dependent intracellular Ca2+ pools have been depleted.

Animals↗
PubMed1994

Simultaneous oscillations of cytoplasmic free Ca2+ concentration and Ins(1,4,5)P3 concentration in mouse pancreatic beta-cells.

Changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in stimulated cells are often oscillatory, but the mechanisms that drive these oscillations are still a matter of controversy: different models of the generation of these [Ca2+]i oscillations make different assumptions as to whether oscillations in Ins(1,4,5)P3 concentration are necessary for this process. We have looked for changes in inositol polyphosphate levels that might occur in suspensions of murine pancreatic beta-cells when these cells are induced to display synchronized oscillations in [Ca2+]i by the sequential addition of glucose, an alpha 2-adrenergic stimulus and extracellular Ca2+. The intracellular level of Ins(1,4,5)P3 oscillated in a manner approximately in synchrony with changes in [Ca2+]i. Oscillations in the levels of Ins(1,4,5)P3 metabolites [Ins(1,3,4)P3 and inositol bisphosphates] were slightly delayed relative to the Ins(1,4,5)P3 oscillations, and the concentration of Ins(1,3,4,5,6)P5 remained approximately constant during the [Ca2+]i oscillations. These results demonstrate that [Ins(1,4,5)P3] and [Ca2+]i oscillate in synchrony in at least one type of cell. Whether such oscillations in intracellular [Ins(1,4,5)P3] provide a primary driving force for [Ca2+]i oscillations either in beta-cells or in other stimulated cells remains to be determined. Even if they do not, the [Ins(1,4,5)P3] oscillations will at least provide an amplifying influence on the [Ca2+]i changes.

Animals↗
PubMed1996

Ins(1,3,4,5)P4 is effective in mobilizing Ca2+ in mouse exocrine pancreatic acinar cells if phospholipase A2 is inhibited.

In enzymically isolated mouse pancreatic acinar cells, under conditions of whole-cell patch-clamp current recording, the effect of phospholipase C-coupled agonists can be mimicked by internal perfusion of the intracellular second messenger Ins(1,4,5)P3 (10 microM) or its analogue Ins(2,4,5)P3 (10 microM). The inositol trisphosphates mimic receptor activation by releasing Ca2+ from intracellular stores and by promoting Ca2+ influx across the surface membrane. This Ca(2+)-mobilizing role of inositol polyphosphates seems to be confined to the inositol trisphosphates because internal perfusion of Ins(1,3,4,5)P4 (10 microM) is not associated with any Ca(2+)-dependent current activation. In this study we investigate the effects of 4-bromophenacyl bromide (4BPB), a putative inhibitor of phospholipase A2 and arachadonic acid production, on inositol polyphosphate-induced Ca2+ signalling. At 10 microM, 4BPB has no effect on unstimulated Ca(2+)-dependent membrane currents. However, if 4BPB is applied to cells internally perfused with 10 microM Ins(1,4,5)P3 or Ins(2,4,5)P3 then the current responses are rapidly potentiated. In cells internally perfused with 10 microM Ins(1,3,4,5)P4, which has itself no effect on membrane currents, application of 4BPB resulted in the activation of Ca(2+)-dependent currents, seen either as repetitive spikes of current or as sustained current activations. The application of arachidonic acid blocks the current responses evoked by the inositol trisphosphates and by Ins(1,3,4,5)P4/4BPB. These results suggest that in enzymically isolated pancreatic acinar cells phospholipase A2 activity is exerting an inhibitory effect on inositol polyphosphate-mediated Ca2+ mobilization. 4BPB removes this inhibition and potentiates the responses to internally perfused inositol trisphosphates and, importantly, makes 10 microM Ins(1,3,4,5)P4 as effective as 10 microM Ins(1,4,5)P3 in mobilizing intracellular Ca2+ and in promoting Ca2+ influx.

Acetophenones↗
PubMed2003

Integrated functional and bioinformatics approach for the identification and experimental verification of RNA signals: application to HIV-1 INS.

Regulation of gene expression involves sequence elements in nucleic acids. In promoters, multiple sequence elements cooperate as functional modules, which in combination determine overall promoter activity. We previously developed computational tools based on this hierarchical structure for in silico promoter analysis. Here we address the functional organization of post-transcriptional control elements, using the HIV-1 genome as a model. Numerous mutagenesis studies demonstrate that expression of HIV structural proteins is restricted by inhibitory sequences (INS) in HIV mRNAs in the absence of the HIV-1 Rev protein. However, previous attempts to detect conserved sequence patterns of HIV-1 INS have failed. We defined four distinct sequence patterns for inhibitory motifs (weight matrices), which identified 22 out of the 25 known INS as well as several new candidate INS regions contained in numerous HIV-1 strains. The conservation of INS motifs within the HIV genome was not due to overall sequence conservation. The functionality of two candidate INS regions was analyzed with a new assay that measures the effect of non-coding mRNA sequences on production of red fluorescent reporter protein. Both new INS regions showed inhibitory activity in sense but not in antisense orientation. Inhibitory activity increased by combining both INS regions in the same mRNA. Inhibitory activity of known and new INS regions was overcome by co-expression of the HIV-1 Rev protein.

Computational Biology↗
PubMed1991

Regulation of smooth muscle contraction in rabbit internal anal sphincter by protein kinase C and Ins(1,4,5)P3.

We have examined the role of protein kinase C (PKC)-beta II and its functional relationship to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and intracellular Ca2+ in the contraction of smooth muscle cells from the rabbit internal and sphincter (IAS). PKC-beta (0.1-100 U/ml) and Ins(1,4,5)P3 (10(-9) to 10(-6) M) caused concentration-dependent contraction of IAS smooth muscle cells permeabilized by saponin. The combination of threshold concentrations of Ins(1,4,5)P3 (10(-9) M) and PKC (0.1 U/ml) was more than additive, causing near maximal shortening (28.2 +/- 2.1% decrease in cell length from control). The response to high concentrations of Ins(1,4,5)P3 and PKC used in combination was not greater than the response to either agent alone. The calmodulin antagonist W-7 (10(-9) M) inhibited the maximal contraction induced by Ins(1,4,5)P3 but not contraction caused by PKC, whereas the PKC antagonist H-7 (10(-6) M) inhibited the maximal contraction induced by PKC but not contraction caused by Ins(1,4,5)P3. Threshold doses of the ionophores A23187 (10(-9) M) and ionomycin (0.2 ng/ml) caused little contraction by themselves, but they potentiated the response elicited by a threshold concentration of PKC (0.1 U/ml), inducing maximal contraction. Preincubation of IAS cells with 4 mM Sr2+, which inhibits the release of intracellular Ca2+, abolished the potentiating effect of Ins(1,4,5)P3 and calcium ionophores on PKC, but the calmodulin antagonist W-7 did not. These data suggest that the contractile effect of maximally effective doses of PKC is independent of the effects of Ins(1,4,5)P3. At submaximal concentrations, however, PKC-dependent contraction is potentiated by Ins(1,4,5)P3 or by ionophore-mediated release of intracellular Ca2+ without requiring calmodulin activation.

Anal Canal↗
PubMed1992

Ruthenium red selectively prevents Ins(1,4,5)P3-but not caffeine-gated calcium release in avian atrium.

We previously reported that inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and caffeine evoked contractures in saponin-permeabilized chick atria. The magnitude of contractures evoked by maximally effective concentrations of Ins(1,4,5)P3 were half those evoked by maximally effective concentrations of caffeine. In the present report, we tested the hypothesis that these two agents may act on distinct calcium-release mechanisms by comparing the effects of ryanodine, ruthenium red, and procaine on the responses to Ins(1,4,5)P3 and caffeine. We find that procaine inhibits both responses with similar mean inhibitory concentrations in the millimolar range. Nanomolar concentrations of ryanodine selectively potentiate the contractures induced by Ins(1,4,5)P3 but have no effect on those induced by caffeine. Micromolar concentrations of ryanodine inhibit responses to both Ins(1,4,5)P3 and caffeine in a use-dependent way. Ruthenium red prevents the response to Ins(1,4,5)P3 and potentiates that to caffeine, as if ruthenium red had enhanced calcium accumulation in the caffeine-sensitive pool(s). Because we found that caffeine prevented the subsequent response to Ins(1,4,5)P3, but Ins(1,4,5)P3 had no detectable effect on the caffeine-induced contracture, we conclude that Ins(1,4,5)P3 and caffeine act on pharmacologically distinct calcium-release mechanisms that may reside in the same sarcoplasmic reticulum compartment.

Actin Cytoskeleton↗
PubMed1999

Pharmacological manipulation of Ins(1,4,5)P3 signaling mimics preconditioning in rabbit heart.

Recent evidence revealed biphasic alterations in myocardial concentrations of the second messenger inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] with ischemic preconditioning (PC), i.e., increase during brief PC ischemia and decrease early during sustained test occlusion. Our aim was to determine whether an agonist and an antagonist of Ins(1,4,5)P(3) signaling (D-myo-inositol-1,4,5-trisphosphate hexasodium salt [D-myo-Ins(1,4, 5)P3] and 2-aminoethoxydiphenyl borate (2-APB), respectively), given such that they mimic this biphasic profile, would mimic infarct size reduction with PC. To test this concept, isolated, buffer-perfused rabbit hearts received no intervention (control), ischemic PC, D-myo-Ins(1,4,5)P3, D-myo-Ins(1,4,5)P(3) + PC, 2-APB, or 2-APB + PC. All hearts then underwent 30-min coronary occlusion and 2 h reflow, and infarct size was delineated by tetrazolium staining. In addition, the effects of D-myo-Ins(1,4,5)P3 and 2-APB on Ins(1,4,5)P3 signaling were evaluated in isolated fura 2-loaded rat cardiomyocytes. Mean infarct size was reduced with PC and in all D-myo-Ins(1,4,5)P3- and 2-APB-treated groups versus control (59 and 42-55%, respectively, vs. 80% of myocardium at risk, P < 0.05). Thus pharmacological manipulation of Ins(1,4,5)P3 signaling mimics the cardioprotection achieved with ischemic PC in rabbit heart.

Animals↗
PubMed2003

Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells.

Rat insulinoma-derived INS-1 cells constitute a widely used beta-cell surrogate. However, due to their nonclonal nature, INS-1 cells are heterogeneous and are not stable over extended culture periods. We have isolated clonal INS-1E cells from parental INS-1 based on both their insulin content and their secretory responses to glucose. Here we describe the stable differentiated INS-1E beta-cell phenotype over 116 passages (no. 27-142) representing a 2.2-yr continuous follow-up. INS-1E cells can be safely cultured and used within passages 40-100 with average insulin contents of 2.30 +/- 0.11 microg/million cells. Glucose-induced insulin secretion was dose-related and similar to rat islet responses. Secretion saturated with a 6.2-fold increase at 15 mm glucose, showing a 50% effective concentration of 10.4 mm. Secretory responses to amino acids and sulfonylurea were similar to those of islets. Moreover, INS-1E cells retained the amplifying pathway, as judged by glucose-evoked augmentation of insulin release in a depolarized state. Regarding metabolic parameters, INS-1E cells exhibited glucose dose-dependent elevations of NAD(P)H, cytosolic Ca(2+), and mitochondrial Ca(2+) levels. In contrast, mitochondrial membrane potential, ATP levels, and cell membrane potential were all fully activated by 7.5 mm glucose. Using the perforated patch clamp technique, 7.5 and 15 mm glucose elicited electrical activity to a similar degree. A K(ATP) current was identified in whole cell voltage clamp using diazoxide and tolbutamide. As in native beta-cells, tolbutamide induced electrical activity, indicating that the K(ATP)conductance is important in setting the resting potential. Therefore, INS-1E cells represent a stable and valuable beta-cell model.

Adenosine Triphosphate↗
PubMed2006

The INS VNTR locus does not associate with smallness for gestational age (SGA) but interacts with SGA to increase insulin resistance in young adults.

CONTEXT: Both adverse intrauterine events and genetic background have been suggested to promote insulin resistance in subjects born small for gestational age (SGA). Among candidate genes that potentially influence both fetal growth and glucose metabolism is insulin. The potential effect of the insulin gene VNTR (INS) on birth weight has been controversial so far. OBJECTIVE: The present association study aimed at testing for the contribution of the INS VNTR locus on birth weight and on the metabolic profile of young adults born SGA (mean age, 22 yr). Two groups of subjects were selected on birth data: SGA (birth weight < 10th percentile; n = 735), and appropriate for gestational age (AGA; birth weight between 25th and 75th percentiles; n = 886). All subjects were genotyped for rs689 A/T single nucleotide polymorphism, in complete linkage disequilibrium with the INS VNTR classes I and III, respectively. RESULTS: Class I INS frequencies were similar in the two groups (70% in AGA; 72% in SGA; P = 0.42). There was significant effect on mean birth weight in neither SGA (P = 0.99) nor AGA (P = 0.18). Although the INS VNTR locus did not associate with anomalies of insulin resistance indices in the AGA group, in the SGA group, INS VNTR class III allele was associated with higher insulin resistance (quantitative insulin sensitivity check index = 0.38 vs. 0.39; P = 0.05). Furthermore, there was evidence of an interaction between the SGA/AGA status and INS VNTR locus on insulin resistance indices (P = 0.01) in a multivariate analysis. CONCLUSION: The INS VNTR locus does not associate in a major way with SGA in the French population. However, our data support an interaction between severe fetal growth restriction and INS VNTR locus, which were associated with insulin resistance in young adults born SGA.

Adult↗
PubMed1995

Nucleus-associated phosphorylation of Ins(1,4,5)P3 to InsP6 in Dictyostelium.

Although many cells contain large amounts of InsP6, its metabolism and function is still largely unknown. In Dictyostelium lysates, the formation of InsP6 by sequential phosphorylation of inositol via Ins(3,4,6)P3 has been described [Stevens and Irvine (1990) Nature (London) 346, 580-583]; the second messenger Ins(1,4,5)P3 was excluded as a potential substrate or intermediate for InsP6 formation. However, we observed that mutant cells labelled in vivo with [3H]inositol showed altered labelling of both [3H]Ins(1,4,5)P3 and [3H]InsP6. In this report we demonstrate that Ins(1,4,5)P3 is converted into InsP6 in vitro by nucleus-associated enzymes, in addition to the previously described stepwise phosphorylation of inositol to InsP6 that occurs in the cytosol. HPLC analysis indicates that Ins(1,4,5)P3 is converted into InsP6 via sequential phosphorylation at the 3-, 6- and 2-positions. Ins[32P]P6, isolated from cells briefly labelled with [32P]Pi, was analysed using Paramecium phytase, which removes the phosphates of InsP6 in a specific sequence. The 6-position contained significantly more 32P radioactivity than the 4- or 5-positions, indicating that the 6-position is phosphorylated after the other two positions. The results from these in vivo and in vitro experiments demonstrate a metabolic route involving the phosphorylation of Ins(1,4,5)P3 via Ins(1,3,4,5)P4 and Ins(1,3,4,5,6)P5 to InsP6 in a nucleus-associated fraction of Dictyostelium cells.

Adenosine Triphosphate↗
PubMed2000

Activation of the Na(+)/H(+) exchanger is required for reperfusion-induced Ins(1,4,5)P(3) generation.

Post-ischemic reperfusion causes a change in inositol phosphate responses to norepinephrine from primary generation of inositol(1,4) bis phosphate (Ins(1,4)P(2)) to generation of inositol(1,4,5) tris phosphate (Ins(1,4,5)P(3)) that is required for the initiation of reperfusion arrhythmias. The current study was undertaken to investigate the role of Na(+)/H(+)exchange in facilitating this transient change in inositol phosphate response. Rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and Ins(1, 4,5)P(3)content was measured by mass analysis or by anion-exchange HPLC following [(3)H]inositol labeling. Reperfusion caused generation of [(3)H]Ins(1,4,5)P(3)(1732+/-398 to 3103+/-214, cpm/g tissue, mean+/-S.E.M., n=5, P<0.01) and the development of arrhythmias. Inhibition of Na(+)/H(+)exchange, by reperfusing at pH 6.3 or by pretreating with HOE-694 (10 n M-3 microM) or HOE-642 (3 microM) prevented the [(3)H]Ins(1,4,5)P(3)generation, without causing any suppression of norepinephrine release. Increases in Ins(1,4,5)P(3)mass were similarly reduced by inhibition of Na(+)/H(+)exchange. Thus, activation of Na(+)/H(+)exchange is required for the enhanced Ins(1,4,5)P(3)response observed under reperfusion conditions, and prevention of Ins(1,4,5)P(3)generation may be an important contributor to the anti-arrhythmic actions of inhibitors of Na(+)/H(+)exchange.

Animals↗
PubMed2005

On the contribution of stereochemistry to human ITPK1 specificity: Ins(1,4,5,6)P4 is not a physiologic substrate.

Ins(1,4,5,6)P4, a biologically active cell constituent, was recently advocated as a substrate of human Ins(3,4,5,6)P4 1-kinase (hITPK1), because stereochemical factors were believed relatively unimportant to specificity [Miller, G.J., Wilson, M.P., Majerus, P.W. and Hurley, J.H. (2005) Specificity determinants in inositol polyphosphate synthesis: crystal structure of inositol 1,3,4-triphosphate 5/6-kinase. Mol. Cell. 18, 201-212]. Contrarily, we provide three examples of hITPK1 stereospecificity. hITPK1 phosphorylates only the 1-hydroxyl of both Ins(3,5,6)P3 and the meso-compound, Ins(4,5,6)P3. Moreover, hITPK1 has >13,000-fold preference for Ins(3,4,5,6)P4 over its enantiomer, Ins(1,4,5,6)P4. The biological significance of hITPK1 being stereospecific, and not physiologically phosphorylating Ins(1,4,5,6)P4, is reinforced by our demonstrating that Ins(1,4,5,6)P4 is phosphorylated (K(m) = 0.18 microM) by inositolphosphate-multikinase.

Humans↗
PubMed1997

Susceptibility of caffeine- and Ins(1,4,5)P3-induced contractions to oxidants in permeabilized vascular smooth muscle.

Two principal pathways of Ca2+ release from the sarcoplasmic reticulum of excitable and non-excitable cells have been described: one pathway dependent on the second messenger D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), and a second pathway sensitive to Ca2+ and regulated by caffeine and ryanodine. It was found that the Ca(2+)-pump activity of vascular smooth muscle sarcoplasmic reticulum is inhibited by superoxide anion radicals (O2.-); however, the effects of reactive oxygen intermediates on sarcoplasmic reticulum Ca2+ release in vascular muscle cells are not well defined. The purpose of the present study was to evaluate the effects of reactive oxygen intermediates generated from the hypoxanthine/xanthine oxidase reaction system on contractions induced by caffeine, Ins(1,4,5)P3 and norepinephrine in staphylococcal alpha-toxin-permeabilized rabbit mesenteric arteries. This system generates O2.-, H2O2, and hydroxyl radicals. We wished to identify which class of reactive oxygen intermediates is responsible for the associated loss of vascular smooth muscle contractile function. Caffeine and Ins(1,4,5)P3 produced a transient contraction when the sarcoplasmic reticulum of the permeabilized, preparations was preloaded with pCa 7.0 solution for 5 min before washing with 0.5 mM EGTA solution; norepinephrine also produced a transient contraction. Exposure of the preparations to hypoxanthine/xanthine oxidase (for 30 min) attenuated caffeine-induced contraction, but was without effect on Ins(1,4,5)P3-induced contraction. The observed effect of hypoxanthine/xanthine oxidase exposure was superoxide dismutase-inhibitable, suggesting O2.- involvement. Hypoxanthine/xanthine oxidase also inhibited norepinephrine-induced contraction. The effect of hypoxanthine/xanthine oxidase on norepinephrine contraction was protected by catalase, but not by superoxide dismutase and dimethyl sulfoxide; exogenously added H2O2 mimicked the effect of hypoxanthine/xanthine oxidase exposure. H2O2, added exogenously, was without effect on Ins(1,4,5)P3-induced contraction. It is suggested that the pathway of Ca2+ release from the sarcoplasmic reticulum dependent on Ins(1,4,5)P3 is insensitive to O2.-. Instead, caffeine-induced Ca2+ release mechanisms may be susceptible to O2.- and H2O2, rather than O2.- and hydroxyl radicals, may be the active agent in the norepinephrine-induced contraction. Our results are also consistent with the view that the attenuation by H2O2 of the norepinephrine-induced contraction may be linked to the receptor-associated pathway of Ins(1,4,5)P3 formation, but not to degradation processes of Ins(1,4,5)P3.

Animals↗
PubMed2002

Regulation of Ins(3,4,5,6)P(4) signaling by a reversible kinase/phosphatase.

Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases.

Calcium↗
PubMed2000

Electrospray ionization mass spectrometric analyses of phospholipids from INS-1 insulinoma cells: comparison to pancreatic islets and effects of fatty acid supplementation on phospholipid composition and insulin secretion.

Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.

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