The digitonin-permeabilized pancreatic islet model. Effect of myo-inositol 1,4,5-trisphosphate on Ca2+ mobilization.
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ABSTRACT:
SUBMITTER: Wolf BA
PROVIDER: S-EPMC1144928 | biostudies-other | 1985 May
REPOSITORIES: biostudies-other
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Project description:In several cell types, including hepatocytes, submaximal concentrations of Ins(1,4,5)P3 stimulate an initial rapid mobilization of intracellular Ca2+ stores that is followed by either no further Ca2+ release or very much slower release. Further additions of Ins(1,4,5)P3 then evoke further Ca2+ mobilization. Such 'incremental' responses [Meyer & Stryer (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3841-3845] could result from all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3 or from partial emptying of stores that are more uniformly sensitive, but unable to release all of their Ca2+ because the response to Ins(1,4,5)P3 rapidly attenuates. By measuring unidirectional 45Ca2+ efflux from intracellular stores stimulated with Ins(1,4,5)P3 under conditions where they continue to sequester 40Ca2+, we provide evidence suggesting that Ins(1,4,5)P3 stimulates all-or-nothing emptying of stores that differ in their sensitivities to Ins(1,4,5)P3, a quantal response pattern.
| S-EPMC1151403 | biostudies-other
Project description:Inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) release intracellular Ca(2+) as localized Ca(2+) signals (Ca(2+) puffs) that represent the activity of small numbers of clustered IP(3)Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca(2+) release channels supporting Ca(2+) puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP(3)R transitions between heterogeneous cellular architectures and the differential behavior of IP(3)Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP(3)Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca(2+) puffs are supported by a broad range of clustered IP(3)R microarchitectures; 2), cluster ultrastructure shapes Ca(2+) puff characteristics; and 3), loosely corralled IP(3)R clusters (>200 nm interchannel separation) fail to coordinate Ca(2+) puffs, owing to inefficient triggering and impaired coupling due to reduced Ca(2+)-induced Ca(2+) release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca(2+) puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca(2+) dynamics.
| S-EPMC3037600 | biostudies-literature
Project description:Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.
| S-EPMC1133688 | biostudies-other
Project description:d-myo-Inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ channels activated by the intracellular messenger inositol 1,4,5-trisphosphate (IP3, 1). The glyconucleotide adenophostin A (AdA, 2) is a potent agonist of IP3Rs. A recent synthesis of d-chiro-inositol adenophostin (InsAdA, 5) employed suitably- protected chiral building blocks and replaced the d-glucose core by d-chiro-inositol. An alternative approach to fully chiral material is now reported using intrinsic sugar chirality to avoid early isomer resolution, involving the coupling of a protected and activated racemic myo-inositol derivative to a d-ribose derivative. Diastereoisomer separation was achieved after trans-isopropylidene group removal, and the absolute ribose-inositol conjugate stereochemistry assigned with reference to the earlier synthesis. Optimization of stannylene-mediated regiospecific benzylation was explored using the model 1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol and conditions successfully transferred to one conjugate diastereoisomer with 3:1 selectivity. However, only roughly 1:1 regiospecificity was achieved on the required diastereoisomer. The regioisomers were successfully separated and transformed subsequently to InsAdA after amination, pan-phosphorylation, and deprotection. InsAdA from this synthetic route bound with greater affinity than AdA to IP3R1 and was more potent in releasing Ca2+ from intracellular stores through IP3Rs. It is the most potent full agonist of IP3R1 and was equipotent with material from the fully chiral synthetic route.
| S-EPMC7116374 | biostudies-literature
Project description:d-myo-Inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ channels activated by the intracellular messenger inositol 1,4,5-trisphosphate (IP3, 1). The glyconucleotide adenophostin A (AdA, 2) is a potent agonist of IP3Rs. A recent synthesis of d-chiro-inositol adenophostin (InsAdA, 5) employed suitably protected chiral building blocks and replaced the d-glucose core by d-chiro-inositol. An alternative approach to fully chiral material is now reported using intrinsic sugar chirality to avoid early isomer resolution, involving the coupling of a protected and activated racemic myo-inositol derivative to a d-ribose derivative. Diastereoisomer separation was achieved after trans-isopropylidene group removal and the absolute ribose-inositol conjugate stereochemistry assigned with reference to the earlier synthesis. Optimization of stannylene-mediated regiospecific benzylation was explored using the model 1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol and conditions successfully transferred to one conjugate diastereoisomer with 3:1 selectivity. However, only roughly 1:1 regiospecificity was achieved on the required diastereoisomer. The conjugate regioisomers of benzyl derivatives 39 and 40 were successfully separated and 39 was transformed subsequently to InsAdA after amination, pan-phosphorylation, and deprotection. InsAdA from this synthetic route bound with greater affinity than AdA to IP3R1 and was more potent in releasing Ca2+ from intracellular stores through IP3Rs. It is the most potent full agonist of IP3R1 known and .equipotent with material from the fully chiral synthetic route.
| S-EPMC7659177 | biostudies-literature
Project description:The effect of the guanine nucleotide GTP on Ca2+ release from the endoplasmic reticulum of digitonin-permeabilized islets was investigated. maximal and half-maximal Ca2+ release were observed at 5 microM- and 2.5 microM-GTP respectively. GTP caused a rapid release of Ca2+ from the endoplasmic reticulum, which was complete within 1 min. GTP-induced Ca2+ release was structurally specific and required the hydrolysis of GTP. The combination of maximal concentrations of GTP (10 microM) and myo-inositol 1,4,5-trisphosphate (IP3) (10 microM) resulted in an additive effect on Ca2+ release from the endoplasmic reticulum. GDP (100 microM), which inhibits GTP-induced Ca2+ release, did not affect IP3-induced Ca2+ release. Furthermore, GTP-induced Ca2+ release was not independent on submicromolar free Ca2+ concentrations, unlike IP3-induced Ca2+ release. These observations suggest that mechanistically GTP-induced Ca2+ release is different from IP3-induced Ca2+ release from the endoplasmic reticulum.
| S-EPMC1147675 | biostudies-other
Project description:Cholesterol depletion reversibly abolishes carbachol-evoked Ca(2+) release from inositol (1,4,5)-trisphosphate (IP3)-sensitive stores, without affecting the distribution of IP3 receptors (IP3R) or endoplasmic reticulum, IP3 formation or responses to photolysis of caged IP3. Receptors that stimulate cAMP formation do not alone evoke Ca(2+) signals, but they potentiate those evoked by carbachol. We show that these potentiated signals are entirely unaffected by cholesterol depletion and that, within individual cells, different IP3-sensitive Ca(2+) stores are released by carbachol alone and by carbachol combined with receptors that stimulate cAMP formation. We suggest that muscarinic acetylcholine receptors in lipid rafts deliver IP3 at high concentration to associated IP3R, stimulating them to release Ca(2+). Muscarinic receptors outside rafts are less closely associated with IP3R and provide insufficient local IP3 to activate IP3R directly. These IP3R, probably type 2 IP3R within a discrete Ca(2+) store, are activated only when their sensitivity is increased by cAMP. Sensitization of IP3R by cAMP extends the effective range of signalling by phospholipase C, allowing muscarinic receptors that are otherwise ineffective to recruit additional IP3-sensitive Ca(2+) stores.
| S-EPMC3672942 | biostudies-literature
Project description:Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP(3)R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2(-/-) mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.
| S-EPMC2881802 | biostudies-literature
Project description:The mobilization of Ca2+ from intracellular stores by Ins(1,4,5)P3 in suspensions of permeabilized rat hepatocytes was potentiated by preincubating intact cells with adenosine 3':5'-cyclic phosphorothioate (cpt-cAMP), or by addition of the catalytic subunit of cyclic-AMP-dependent protein kinase (PKA) after cell permeabilization. This action of PKA involved both an enhancement in Ins(1,4,5)P3 sensitivity and an increase in the size of the Ins(1,4,5)P3-releasable Ca2+ pool. Inclusion of the protein phosphatase inhibitor okadaic acid in the permeabilization medium augmented the effects of PKA. Treatment with PKA catalytic subunit also increased the rate of ATP-dependent Ca2+ sequestration. To determine whether the effects of PKA on the Ca(2+)-release mechanism were secondary to alterations in the Ca2+ load of the Ins(1,4,5)P3-sensitive stores, a method was developed using Mn2+ as a Ca2+ surrogate to examine the permeability properties of the Ins(1,4,5)P3-gated channels independent of Ca2+ fluxes. This approach utilized the ability of Mn2+ to quench the fluorescence of fura-2 compartmentalized within intracellular Ca2+ stores in an Ins(1,4,5)P3-dependent manner, with thapsigargin added to block the ATP-activated Ca2+ pump and to ensure that the Ca2+ stores were fully depleted of Ca2+. The initial rate and extent of Mn2+ quenching of compartmentalized fura-2 was increased in a dose-dependent manner by Ins(1,4,5)P3. PKA activation increased both the initial rate and the extent of Mn2+ quenching at sub-maximal Ins(1,4,5)P3 doses, but there was no effect on the quench rate in the presence of saturating Ins(1,4,5)P3. However, the amount of compartmentalized fura-2 that could be quenched by Mn2+ in the presence of maximal Ins(1,4,5)P3 was increased by PKA. These data suggest two distinct actions of PKA on the Ins(1,4,5)P3-sensitive Ca2+ stores. (1) Modification of the ion-permeability properties of the Ins(1,4,5)P3 receptor/channel through an increase in the sensitivity to Ins(1,4,5)P3 for channel opening. (2) A recruitment of Ca2+ stores from the Ins(1,4,5)P3-insensitive pool. Both actions were independent of the Ca(2+)-loading state of the stores. Imaging studies of single permeabilized hepatocytes showed that the Ins(1,4,5)P3-sensitive stores were distributed throughout the cell and PKA enhanced the rate of Ins(1,4,5)P3-stimulated Mn2+ quench in individual cells, without modifying the subcellular distribution of Ins(1,4,5)P3-sensitive stores.
| S-EPMC1134376 | biostudies-other
Project description:AIMS:Phospholamban (PLB) is the key regulator of the cardiac Ca2+ pump (SERCA2a)-mediated sarcoplasmic reticulum Ca2+ stores. We recently reported that PLB is highly concentrated in the nuclear envelope (NE) from where it can modulate perinuclear Ca2+ handling of the cardiomyocytes (CMs). Since inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) mediates nuclear Ca2+ release, we examined whether the nuclear pool of PLB regulates IP3-induced nuclear Ca2+ handling. METHODS AND RESULTS:Fluo-4 based confocal Ca2+ imaging was performed to measure Ca2+ dynamics across both nucleus and cytosol in saponin-permeabilized CMs isolated from wild-type (WT) or PLB-knockout (PLB-KO) mice. At diastolic intracellular Ca2+ ([Ca2+]i?=?100?nM), the Fab fragment of the monoclonal PLB antibody (anti-PLB Fab) facilitated the formation and increased the length of spontaneous Ca2+ waves (SCWs) originating from the nuclear region in CMs from WT but not from PLB-KO mice. We next examined nuclear Ca2+ activities at basal condition and after sequential addition of IP3, anti-PLB Fab, and the IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) at a series of [Ca2+]i. In WT mice, at 10?nM [Ca2+]i where ryanodine receptor (RyR2) based spontaneous Ca2+ sparks rarely occurred, IP3 increased fluorescence amplitude (F/F0) of overall nuclear region to 1.19?±?0.02. Subsequent addition of anti-PLB Fab significantly decreased F/F0 to 1.09?±?0.02. At 50?nM [Ca2+]i, anti-PLB Fab not only decreased the overall nuclear F/F0 previously elevated by IP3, but also increased the amplitude and duration of spark-like nuclear Ca2+ release events. These nuclear Ca2+ releases were blocked by 2-APB. At 100?nM [Ca2+]i, IP3 induced short SCWs originating from nucleus. Anti-PLB Fab transformed those short waves into long SCWs with propagation from the nucleus into the cytosol. In contrast, neither nuclear nor cytosolic Ca2+ dynamics was affected by anti-PLB Fab in CMs from PLB-KO mice in all these conditions. Furthermore, in WT CMs pretreated with RyR2 blocker tetracaine, IP3 and anti-PLB Fab still increased the magnitude of nuclear Ca2+ release but failed to regenerate SCWs. Finally, anti-PLB Fab increased low Ca2+ affinity mag-fluo 4 fluorescence intensity in the lumen of NE of nuclei isolated from WT but not in PLB-KO mice. CONCLUSION:PLB regulates nuclear Ca2+ handling. By increasing Ca2+ uptake into lumen of the NE and perhaps other perinuclear membranes, the acute reversal of PLB inhibition decreases global Ca2+ concentration at rest in the nucleoplasm, and increases Ca2+ release into the nucleus, through mechanisms involving IP3R and RyR2 in the vicinity.
| S-EPMC6205300 | biostudies-literature