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B V Potter

Publications and source records attributed to B V Potter.

At least 127 records · Page 7Linked to original sources
PubMed1991

Inositol triphosphate produces different patterns of cytoplasmic Ca2+ spiking depending on its concentration.

In single mouse pancreatic acinar cells the effects of intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or the non-metabolizable InsP3 analogue inositol 1,4,5-triphosphorothioate (InsPS3) have been investigated using a wide range of concentrations. Different types of cytosolic Ca2+ fluctuation patterns (monitored as Ca(2+)-dependent Cl- current in patch-clamp whole-cell recording experiments) could be generated by InsP3 or InsPS3, dependent on concentration, resembling those previously shown to be evoked by varying degrees of receptor activation in these cells. Low InsPS3 concentrations evoked repetitive local Ca2+ spikes whereas at relatively high concentrations repetitive Ca2+ waves were produced. In the presence of intracellular citrate a much lower messenger level was sufficient to generate waves. The InsP3 concentration determines whether the cytosolic Ca2+ signals are local or global.

Animals↗
PubMed1991

Mobilization of calcium by inositol trisphosphates from permeabilized rat parotid acinar cells. Evidence for translocation of calcium from uptake to release sites within the inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pool.

D-myo-Inositol (1,4,5)-trisphosphate ((1,4,5)IP3)-induced Ca2+ release and subsequent Ca2+ reuptake were investigated in saponin-permeabilized rat parotid acinar cells. Following the rapid release of Ca2+ by (1,4,5)IP3, Ca2+ was resequestered. The sequential addition of submaximal concentrations of (1,4,5)IP3 resulted in sequential Ca2+ release. However, when the cells were challenged with the poorly metabolized (1,4,5)IP3 analogues, (1,4,5)IPS3 or (2,4,5)IP3, or under conditions where the metabolism of authentic (1,4,5)IP3 was reduced, Ca2+ reuptake again occurred, but sequestered Ca2+ was not released by subsequent additions of (1,4,5)IP3. The sequestered Ca2+ was, however, released by thapsigargin, an agent which inhibits active Ca2+ uptake into the (1,4,5)IP3-sensitive pool. Furthermore, the rate of thapsigargin-induced release was significantly increased in the continued presence of an (1,4,5)IP3 stimulus. Thus, Ca2+ reuptake apparently occurred into the (1,4,5)IP3- and thapsigargin-sensitive Ca2+ store and (1,4,5)IP3 continued to influence the permeability of this pool to Ca2+ during Ca2+ reuptake. In contrast to the findings in permeabilized cells, Ca2+ reuptake did not occur in the sustained presence of (1,4,5)IP3 in intact parotid cells. We conclude that cell permeabilization reveals a kinetic, and presumably structural, separation of Ca2+ uptake and release sites within the (1,4,5)IP3-regulated intracellular organelle.

Animals↗
PubMed1991

Interactions between inositol tris- and tetrakis-phosphates. Effects on intracellular Ca2+ mobilization in SH-SY5Y cells.

The potential Ca2(+)-releasing activity of the inositol tetrakisphosphates Ins(1,3,4,6)P4 and DL-Ins(1,4,5,6)P4 and the inositol pentakisphosphate Ins(1,3,4,5,6)P5 and their effect on Ins(1,4,5)P3- and DL-Ins (1,3,4,5)P4-mediated Ca2+ release were examined in permeabilized SH-SY5Y human neuroblastoma cells. Neither DL-Ins(1,4,5,6)P4 nor Ins(1,3,4,5,6)P5 exhibit Ca2(+)-releasing activity at concentrations up to 10 microM, but Ins(1,3,4,6)P4 releases Ca2+ dose-dependently, with an EC50 value (conen, giving half-maximal effect) of 5.92 +/- 0.47 microM. Maximal response by this tetrakisphosphate (49 +/- 2.5%) is significantly less than that seen with Ins(1,4,5)P3 (60 +/- 3%) and is achieved at a concentration of 30 microM. In the presence of this concentration of Ins(1,3,4,6)P4 the EC50 value for Ins(1,4,5)P3-mediated Ca2+ release increases from 0.12 +/- 0.02 microM to 2.11 +/- 0.51 microM, providing evidence that this naturally occurring inositol tetrakisphosphate may recognize and exhibit its Ca2(+)-releasing activity via the Ins(1,4,5)P3 receptor. DL-Ins(1,3,4,5)P4, however, at its maximally effective concentration (10 microM) does not significantly affect Ins(1,4,5)P3-mediated Ca2+ release, and therefore appears to mediate its Ca2(+)-mobilizing action through a receptor distinct from that for Ins(1,4,5)P3.

Binding, Competitive↗
PubMed1991

D-[35S(U)]inositol 1,4,5-trisphosphorothioate, a novel radioligand for the inositol 1,4,5-trisphosphate receptor. Complex binding to rat cerebellar membranes.

D-[35S(U)]myo-inositol 1,4,5-trisphosphorothioate [( 35S]InsPS3), a synthetic, metabolically stable analogue of inositol 1,4,5-trisphosphate (InsP3), binds with high affinity (Kd 58.6 +/- 9.1 nM) to rat cerebellar membranes revealing a high density of specific binding sites (Bmax 21.5 +/- 2.1 pmol/mg of protein). Comparison with [3H]InsP3 binding reveals a higher density of sites labelled by [35S]InsPS3 and complex competition curves for displacement of specific [35S]InsPS3 by InsP3. The results suggest that [35S]InsPS3 labels two sites in rat cerebellar membranes with equal affinity: the InsP3 receptor and a site that displays low affinity for InsP3.

Animals↗
PubMed1991

Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum.

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude 'P2' cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

Animals↗
PubMed1991

Interaction of synthetic D-6-deoxy-myo-inositol 1,4,5-trisphosphate with the Ca2(+)-releasing D-myo-inositol 1,4,5-trisphosphate receptor, and the metabolic enzymes 5-phosphatase and 3-kinase.

The ability of D-6-deoxy-myo-inositol 1,4,5-trisphosphate [6-deoxy-Ins(1,4,5)P3], a synthetic analogue of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to mobilise intracellular Ca2+ stores in permeabilised SH-SY5Y neuroblastoma cells was investigated. 6-Deoxy-Ins(1,4,5)P3 was a full agonist (EC50 = 6.4 microM), but was some 70-fold less potent than Ins (1,4,5)P3 (EC50 = 0.09 microM), indicating that the 6-hydroxyl group of Ins(1,4,5)P3 is important for receptor binding and stimulation of Ca2+ release, but is not an essential structural feature. 6-Deoxy-Ins(1,4,5)P3 was not a substrate for Ins (1,4,5)P3 5-phosphatase, but inhibited both the hydrolysis of 5-[32P]+ Ins (1,4,5)P3 (Ki 76 microM) and the phosphorylation of [3H]Ins(1,4,5)P3 (apparent Ki 5.7 microM). 6-Deoxy-Ins (1,4,5)P3 mobilized Ca2+ with different kinetics to Ins(1,4,5)P3, indicating that it is probably a substrate for Ins (1,4,5)P3 3-kinase.

Animals↗
PubMed1991

Influences of separation and adjacent sequences on the use of alternative 5' splice sites.

Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.

Animals↗
PubMed1991

Injection of inositol trisphosphorothioate into Limulus ventral photoreceptors causes oscillations of free cytosolic calcium.

Limulus ventral photoreceptors contain calcium stores sensitive to release by D-myo-inositol 1,4,5 trisphosphate (InsP3) and a calcium-activated conductance that depolarizes the cell. Mechanisms that terminate the response to InsP3 were investigated using nonmetabolizable DL-myo-inositol 1,4,5 trisphosphorothioate (InsPS3). An injection of 1 mM InsPS3 into a photoreceptor's light-sensitive lobe caused an initial elevation of cytosolic free calcium ion concentration (Cai) and a depolarization lasting only 1-2 s. A period of densensitization followed, during which injections of InsPS3 were ineffective. As sensitivity recovered, oscillations of membrane potential began, continuing for many minutes with a frequency of 0.07-0.3 Hz. The activity of InsPS3 probably results from the D-stereoisomer, since L-InsP3 was much less effective than InsP3. Injections of 1 mM InsP3 caused an initial depolarization and a period of densensitization similar to that caused by 1 mM InsPS3, but no sustained oscillations of membrane potential. The initial response to InsPS3 or InsP3 may therefore be terminated by densensitization, rather than by metabolism. Metabolism of InsP3 may prevent oscillations of membrane potential after sensitivity has recovered. The InsPS3-induced oscillations of membrane potential accompanied oscillations of Cai and were abolished by injection of ethyleneglycol-bis (beta-aminoethyl ether)-N,N'-tetraacetic acid. Removal of extracellular calcium reduced the frequency of oscillation but not its amplitude. Under voltage clamp, oscillations of inward current were observed. These results indicate that periodic bursts of calcium release underly the oscillations of membrane potential. After each burst, the sensitivity of the cell to injected InsP3 was greatly reduced, recovering during the interburst interval. The oscillations may, therefore, result in part from a periodic variation in sensitivity to a constant concentration of InsPS3. Prior injection of calcium inhibited depolarization by InsPS3, suggesting that feedback inhibition of InsPS3-induced calcium release by elevated Cai may mediate desensitization between bursts and after injections of InsPS3.

Animals↗
PubMed1991

Synthetic phosphorothioate-containing analogues of inositol 1,4,5-trisphosphate mobilize intracellular Ca2+ stores and interact differentially with inositol 1,4,5-trisphosphate 5-phosphatase and 3-kinase.

Intracellular Ca2+ stores in permeabilized SH-SY5Y neuroblastoma cells were mobilized by D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P3] and two of its synthetic analogues, DL-myo-inositol 1,4-bisphosphate 5-phosphorothioate (DL-InsP3-5S) and DL-myo-inositol 1,4,5-trisphosphorothioate (DL-InsP3S3). The concentrations of D-Ins(1,4,5)P3, DL-InsP3-5S, and DL-InsP3S3 required for half-maximal release were 0.11, 0.8, and 2.5 microM, respectively. All agents were full agonists, releasing 55-60% of sequestered 45Ca2+. D-Ins(1,4,5)P3-induced mobilization of Ca2+ was transient, and Ca2+ reuptake followed D-Ins(1,4,5)P3 metabolism closely. DL-InsP3S3-induced mobilization was persistent, consistent with the resistance of this analogue to metabolic enzymes. In contrast, DL-InsP3-5S-induced Ca2+ mobilization was followed by reuptake of Ca2+, albeit at a slower rate than that seen with D-Ins(1,4,5)P3. DL-InsP3-5S and DL-InsP3S3 were resistant to D-Ins(1,4,5)P3 5-phosphatase and potently inhibited the enzyme, with Ki values of 6.8 and 1.7 microM, respectively. DL-InsP3S3 was resistant to D-Ins(1,4,5)P3 3-kinase and was a very weak inhibitor of the enzyme (Ki = 230 microM). The ability of DL-InsP3-5S to inhibit D-Ins(1,4,5)P3 phosphorylation (apparent Ki = 5 microM) and its loss of Ca(2+)-releasing ability on incubation with D-Ins(1,4,5)P3 3-kinase suggest that this analogue may undergo phosphorylation to inositol 1,3,4-trisphosphate 5-phosphorothioate. These differential and complementary properties of DL-InsP3-5S and DL-InsP3S3 may be useful in dissecting the roles of D-Ins(1,4,5)P3 and D-myo-inositol 1,3,4,5-tetrakisphosphate in Ca2+ homeostasis.

Calcium↗
PubMed1991

The effect of inositol 1,4,5-trisphosphate and inositol 1,4,5-trisphosphorothioate on calcium release and membrane skeleton organization in the human red blood cell.

The inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] content of unstimulated human red blood cells (RBC) has been found to be 227 +/- 83 pmol of Ins(1,4,5)P3 per mL of packed cells. Ins (1,4,5)P3 at concentrations between 0.4-5.0 microM releases Ca2+ from RBC plasma membrane with an EC50 of 0.55 microM. Inositol 4,5-bisphosphate [Ins(4,5)P2] is less potent, but inositol 1-phosphate [Ins(1)P], inositol 1,4-bisphosphate [Ins (1,4)P2], and adenosine triphosphate (ATP) are inactive. The release was stereospecific for D-Ins(1,4,5)P3; 1 microM L-Ins(1,4,5)P3 released no more Ca2+ than the control. A nonhydrolyzable analog of Ins(1,4,5)P3, inositol 1,4,5-trisphosphorothioate [Ins(1,4,5)PS3] evokes sustained release of Ca2+ from isolated ghosts. Release of 45Ca2+ was also observed after the addition of AlF4-. Ionophore A23187 and AlF4- increase the level of Ins(1,4,5)P3 in intact RBC to 1004 +/- 533 and 455 +/- 74 pmol/mL of packed RBC, respectively. We have elaborated a method for visualization of spectrin by indirect immunofluorescence in smears of RBC. Ins(1,4,5)P3 evokes shape changes in permeabilized RBC and disorganization of the spectrin network. The shape changes are stereospecific for the D-enantiomer, since L-Ins(1,4,5)P3 and other compounds had no effect. Whereas the effect of Ins(1,4,5)P3 was reversible, as was the weaker effect of Ins(4,5)P2, Ins(1,4,5)PS3 evoked irreversible shape changes. Shape changes and spectrin disorganization were also observed after the action of AlF4- and ionophore A23187. We conclude that the phosphoinositide signaling system plays an important role in the shape maintenance of human RBC.

Aluminum↗
PubMed1990

Ca2(+)-mobilising properties of synthetic fluoro-analogues of myo-inositol 1,4,5-trisphosphate and their interaction with myo-inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

The ability of two fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. DL-2-deoxy-2-fluoro-scyllo-Ins(1,4,5)P3 (2F-Ins(1,4,5)P3) and DL-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 (2,2-F2-Ins(1,4,5)P3) were full agonists (EC50s 0.77 and 0.41 microM respectively) and slightly less potent than D-Ins(1,4,5)P3 (EC50 0.13 microM), indicating that the axial 2-hydroxyl group of Ins(1,4,5)P3 is relatively unimportant in receptor binding and stimulation of Ca2+ release. Both analogues mobilized Ca2+ with broadly similar kinetics and were substrates for Ins(1,4,5)P3 3-kinase but, qualitatively, were slightly poorer than Ins(1,4,5)P3. 2F-Ins(1,4,5)P3 was a weak substrate for Ins(1,4,5)P3 5-phosphatase but 2,2-F2-Ins(1,4,5)P3 was apparently not hydrolysed by this enzyme, although it inhibited its activity potently (Ki = 26 microM).

Animals↗
PubMed1990

Inositol 1,3,4,5-tetrakisphosphate-induced release of intracellular Ca2+ in SH-SY5Y neuroblastoma cells.

Inositol-polyphosphate-induced Ca2+ mobilization was investigated in saponin-permeabilized SH-SY5Y human neuroblastoma cells. Ins(1,4,5)P3 induced a dose-related release from intracellular Ca2+ stores with an EC50 (concn. giving half-maximal effect) of 0.1 microM and a maximal release of 70%. Ins(1,3,4)P3, DL-Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5 did not evoke Ca2+ mobilization in these cells when used at concentrations up to 10 microM. However, Ins(1,3,4,5)P4 was found to release Ca2+ in a dose-related manner, but the response was dependent on the source of Ins(1,3,4,5)P4 used. When commercially available D-Ins(1,3,4,5)P4 was used, the EC50 and maximal response values were 1 microM and 50% respectively, compared with values for chemically synthesized DL-Ins(1,3,4,5)P4 of 2 microM and 25%. The enhanced maximal response of commercial D-Ins(1,3,4,5)P4 was decreased by pretreatment with rat brain crude Ins(1,4,5)P3 3-kinase and was therefore concluded to be indicative of initial Ins(1,4,5)P3 contamination of the Ins(1,3,4,5)P4 preparation. When metabolism of DL-Ins(1,3,4,5)P4 (10 microM) in these cells at 25 degrees C was investigated by h.p.l.c., substantial amounts of Ins(1,4,5)P3 (0.2 microM) and Ins(1,3,4)P3 (0.8 microM) were found to be produced within 3 min. Analysis of DL-Ins(1,3,4,5)P4 incubation with cells at 4 degrees C, however, indicated that metabolism had been arrested ([3H]Ins(1,4,5)P3 detection limits were estimated to be approx. 0.01 microM). When chemically synthesized DL-Ins(1,3,4,5)P4 and incubation conditions of low temperature were used, the Ca2(+)-releasing properties of this compound were established to be 1 microM and 19% for the EC50 and maximal response values respectively. The results obtained strongly suggest that Ins(1,3,4,5)P4 alone has the ability to release intracellular Ca2+. However, in the presence of sub-maximal concentrations of Ins(1,4,5)P3, Ca2+ release appears to be synergistic with Ins(1,3,4,5)P4, but at supramaximal concentrations not even additive effects are observed.

Calcium↗
PubMed1990

Inhibition of inositol 1,4,5-trisphosphate metabolism in permeabilised SH-SY5Y human neuroblastoma cells by a phosphorothioate-containing analogue of inositol 1,4,5-trisphosphate.

Electrically permeabilised [3H]inositol-labelled SH-SY5Y human neuroblastoma cells were employed to examine the effects of two synthetic, phosphatase-resistant analogues of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on the metabolism of cell membrane-derived [3H]Ins(1,4,5)P3 or exogenous [5-32P]Ins(1,4,4)P3. Incubation of permeabilised SH-SY5Y cells for 5 min at 37 degrees C with carbachol and guanosine 5'-[gamma-thio]triphosphate caused a decrease in [3H]phosphoinositol phospholipid levels and an increase in [3H]inositol phosphate accumulation with inositol 4-phosphate, inositol 1,4-bisphosphate, Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate comprising approximately 79%, 16%, 3% and 2%, respectively, of the increase. Inositol 1-phosphate levels did not increase upon stimulation, nor was inositol 4-phosphate converted rapidly to inositol. In parallel incubations, the analogues, DL-inositol 1,4,5-trisphosphorothioate (DL-InsP3S3) and DL-inositol 1,4-bisphosphate 5-phosphorothioate (DL-InsP3S), and synthetic racemic Ins(1,4,5)P3 (DL-InsP3), altered the profile of the [3H]inositol phosphates recovered and led, at millimolar concentrations, to a 10-15-fold increase in [3H]Ins(1,4,5)P3. The extent of inhibition of [3H]Ins(1,4,5)P3 metabolism was, however, greatest in the presence of synthetic D-Ins(1,4,5)P3 (greater than or equal to 5 mM), when [3H]Ins(1,4,5)P3 comprised approximately 50% of the increase in total [3H]inositol phosphates. Thus, under these conditions, at least 50% of [3H]inositol phosphates were derived from [3H]phosphatidylinositol 4,5-bisphosphate. [32P]Pi release from exogenous [5-32P]Ins(1,4,5)P3 was also inhibited by DL-InsP3S3, DL-InsP3S and DL-InsP3, with half-maximal inhibition at approximately 50 microM, 160 microM and 240 microM respectively. These actions were approximately ten times more potent than the effects of these compounds on [3H]inositol phosphate accumulation, indicating that homogenous mixing of exogenous and cell-membrane-derived Ins(1,4,5)P3 does not occur. These findings indicate that DL-InsP3S3 and DL-InsP3S inhibit Ins(1,4,5)P3 5-phosphatase. In contrast, the effects of synthetic DL-InsP3 and D-Ins(1,4,5)P3 are due to isotopic dilution. Whilst DL-InsP3S3 was the most potent inhibitor of dephosphorylation of exogenous or cell-membrane-derived Ins(1,4,5)P3, it was the weakest inhibitor of 3-kinase-catalysed Ins(1,4,5)P3 phosphorylation. Similarly, although approximately 50 times less potent than DL-InsP3S3, 2,3-diphosphoglycerate inhibited Ins(1,4,5)P3 5-phosphatase activity and was apparently without effect of Ins(1,4,5)P3 3-kinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Effect of inositol trisphosphate and calcium on oscillating elevations of intracellular calcium in Xenopus oocytes.

Stimulation of many nonexcitable cells by Ca2(+)-mobilizing receptor agonists causes oscillating elevations of the intracellular free Ca2+ concentration ((Ca2+]i), rather than a continuous increase. It has been proposed that the frequency at which [Ca2+]i oscillates determines the biological response. Because the occurrence of [Ca2+] oscillations is observed together with endogenous inositol polyphosphate (InsPs) production or following InsPs application, we injected Xenopus laevis oocytes with InsPs and monitored Ca2(+)-activated Cl- currents as an assay of [Ca2+]i. Microinjection of the poorly metabolizable inositol trisphosphate (InsP3) derivatives inositol 2,4,5-trisphosphate (Ins(2,4,5)P3) and inositol 1,4,5-trisphosphorothioate (Ins(1,4,5) P3S3) induced [Ca2+]i oscillations. The frequency at which [Ca2+]i oscillated increased with the injected dose, indicating that the frequency-generating mechanism lies distal to InsP3 production and that generation of oscillations does not require either oscillation of InsP3 levels or InsP3 metabolism. Injections of high doses of Ins(1,4,5)P3 or Ins(2,4,5)P3 inhibited ongoing oscillations, whereas Ca2+ injections decreased the amplitude of Ins(2,4,5)P3-induced oscillations without altering their frequency. Injections of the Ins(1,4,5)P3 metabolite inositol 1,3,4,5-tetrakisphosphate also caused oscillations whose frequency was related to the injected dose, although inositol tetrakisphosphate injection induced an increase in the cellular level of Ins(1,4,5)P3. The results suggest a multicomponent oscillatory system that includes the InsP3 target as well as a Ca2(+)-sensitive step that modulates amplitude.

Acetylcholine↗
PubMed1990

The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.

Adenosine Triphosphate↗