The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.
ABSTRACT: 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.
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.
Project description:Depletion of the Ins(1,4,5)P3-sensitive intracellular Ca2+ store of vascular endothelial cells after selective inhibition of the endoplasmic-reticulum (ER) Ca2+ pump by thapsigargin or 2,5-di-t-butylhydroquinone (BHQ) increases Ca2+ influx from the extracellular space in the absence of phosphoinositide hydrolysis. One model to account for these results suggests a close association between the internal store and the plasmalemma, allowing for the vectorial movement of Ca2+ from the extracellular space to the ER. Furthermore, recent evidence suggests that Ins(1,4,5)P3-induced Ca2+ release from intracellular stores is regulated by the free cytosolic Ca2+ concentration ([Ca2+]i). Thus agonist-induced Ca2+ entry may directly regulate Ca2+ release from internal stores. To test these hypotheses, we examined the effect of 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SKF 96365), an inhibitor of Ca2+ influx, on unidirectional 45Ca2+ efflux (i.e. retrograde radioisotope flux via the influx pathway) and on [Ca2+]i as measured by fura-2. Bradykinin produced a transient increase in [Ca2+]i, reflecting release of Ca2+ from internal stores, and a sustained increase indicative of Ca2+ influx. In the absence of agonist, 45Ca2+ efflux was slow and monoexponential with time. Addition of BK dramatically increased 45Ca2+ efflux; 50-60% of the 45Ca2+ associated with the cell monolayer was released within 2 min after addition of bradykinin. Both the bradykinin-induced change in [Ca2+]i and the stimulation of 45Ca2+ efflux was completely blocked by loading the cells with the Ca2+ chelator BAPTA. At a supermaximal concentration of bradykinin (50 nM), SKF 96365 (50 microM) inhibited the rise in [Ca2+]i attributed to influx without affecting release from internal stores. At a threshold concentration of bradykinin (2 nM), SKF 96365 blocked influx, but stimulated Ca2+ release from internal stores, as indicated by increases in both the transient component of the fura-2 response and 45Ca2+ efflux. Thapsigargin (200 nM) and BHQ (10 microM) produced an increase in 45Ca2+ efflux that was completely blocked by SKF 96365 or by cytosolic loading with BAPTA. These results suggest the existence of a restricted sub-plasmalemmal space that is defined by an area of surface membrane which contains the Ca(2+)-influx pathway but is devoid of Ca2+ pumps, and by a section of ER that is rich in thapsigargin-sensitive Ca(2+)-pump units.(ABSTRACT TRUNCATED AT 400 WORDS)
Project description:D-Ins(1,4,5)P3 is now recognized as an intracellular messenger that mediates the actions of many cell-surface receptors on intracellular Ca2+ pools, but its complex and rapid metabolism in intact cells has confused interpretation of its possible roles in oscillatory changes in intracellular [Ca2+] and in controlling Ca2+ entry at the plasma membrane. We now report the actions and metabolic stability of a synthetic analogue of Ins(1,4,5)P3, DL-inositol 1,4,5-trisphosphorothioate [DL-Ins(1,4,5)P3[S]3]. In permeabilized hepatocytes, DL-Ins(1,4,5)P3[S]3 and synthetic DL-Ins(1,4,5)P3 stimulated Ca2+ release from the same intracellular stores, though the concentration required for half-maximal release was 3-fold higher for DL-Ins(1,4,5)P3[S]3. Since L-Ins(1,4,5)P3 neither antagonized the effects of D-Ins(1,4,5)P3 nor itself stimulated appreciable Ca2+ release, the activity of the racemic mixture of Ins(1,4,5)P3, and presumably also of Ins(1,4,5)P3[S]3, is attributable to the D-isomer. Under conditions where there was negligible metabolism of D-[3H]Ins(1,4,5)P3, both DL-Ins(1,4,5)P3 and DL-Ins(1,4,5)P3[S]3 elicited rapid Ca2+ release from intracellular stores, and the stores remained empty during prolonged stimulation. When cells were incubated at high density, both compounds stimulated rapid Ca2+ release, but while the stores soon refilled as Ins(1,4,5)P3 was degraded to Ins(1,4)P2, there was no refilling of the pools after stimulation with DL-Ins(1,4,5)P3[S]3. When DL-Ins(1,4,5)P3 or DL-Ins(1,4,5)P3[S]3 was treated with a crude preparation of Ins(1,4,5)P3 3-kinase and ATP, and the Ca2+-releasing activity of the products subsequently assayed, DL-Ins(1,4,5)P3 was completely inactivated by phosphorylation, but there was no loss of activity of the phosphorothioate analogue. In additional experiments, DL-Ins(1,4,5)P3[S]3 (10 microM) did not affect the rate of phosphorylation of D-[3H]Ins(1,4,5)P3 (1 microM). We conclude that Ins(1,4,5)P3[S]3 is a full agonist and only 3-fold less potent than Ins(1,4,5)P3 in mobilizing intracellular Ca2+ stores, but unlike the natural messenger it is resistant to both phosphorylation and dephosphorylation. We propose that this stable analogue will allow the direct actions of Ins(1,4,5)P3 to be resolved from those that require its metabolism.
Project description:Ins(2,4,5)P3, a metabolically stable analogue of Ins(1,4,5)P3, is widely used in analyses of Ca2+ signalling pathways, but its utility depends upon it faithfully mimicking the effects of the natural messenger, Ins(1,4,5)P3, at InsP3 receptors. To compare the kinetics of InsP3-evoked 45Ca2+ mobilization, Ins(1,4,5)P3- and Ins(2,4,5)P3-stimulated 45Ca2+ release from the intracellular stores of permeabilized rat hepatocytes was measured using rapid superfusion. Both Ins(1,4,5)P3 and Ins(2,4,5)P3 caused concentration-dependent increases in the rate of 45Ca2+ efflux, which accelerated towards a peak and then abruptly switched to a bi-exponentially decaying release rate. However, the peak rate of 45Ca2+ mobilization evoked by maximal concentrations of Ins(2,4,5)P3 was only 65+/-3% (n = 3) of that evoked by Ins(1,4,5)P3. Furthermore, Ins(2,4,5)P3 inhibited the peak rate of 45Ca2+ efflux evoked by Ins(1,4,5)P3. These results indicate that Ins(2,4,5)P3 is a partial agonist at hepatic Ins(1,4,5)P3 receptors. Additionally, responses to Ins(2,4,5)P3 were less positively cooperative [Hill coefficient (h) = 1.9+/-0.3] than were those to Ins(1,4,5)P3 (h = 3.0+/-0.2) and the kinetics of termination of 45Ca2+ mobilization were slower. The lesser efficacy of Ins(2,4,5)P3 may account for the lower cooperativity in the responses it evokes, the slower inactivation of InsP3 receptors and the characteristic patterns of Ca2+ spiking it evokes in intact cells.
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.
Project description:Ins(1,4,5)P3 is the intracellular messenger that mediates the effects of many cell-surface receptors on intracellular Ca2+ stores. Although radioligand-binding studies have identified high-affinity Ins(1,4,5)P3-binding sites in many tissues, these have not yet been convincingly shown to be the receptors that mediate Ca2+ mobilization, nor is it clear whether there are differences in these binding sites between tissues. Here we report that Ins(1,4,5)P3 binds to a single class of high-affinity sites in both permeabilized hepatocytes (KD = 7.8 +/- 1.1 nM) and cerebellar membranes (KD = 6.5 +/- 2.4 nM), and provide evidence that these are unlikely to reflect binding to either of the enzymes known to metabolize Ins(1,4,5)P3. Furthermore, the rank order of potency of synthetic inositol phosphate analogues in displacing specifically bound Ins(1,4,5)P3 is the same as their rank order of potency in stimulating mobilization of intracellular Ca2+ stores, suggesting that the Ins(1,4,5)P3-binding site may be the physiological receptor. Radiation inactivation of the Ins(1,4,5)P3-binding sites of liver and cerebellum reveals that they have similar molecular target sizes: 257 +/- 36 kDa in liver and 258 +/- 20 kDa in cerebellum. We conclude that an Ins(1,4,5)P3-binding protein with a molecular target size of about 260 kDa is probably the receptor that mediates Ca2+ mobilization in hepatocytes, and our limited data provide no evidence to distinguish this from the cerebellar Ins(1,4,5)P3-binding protein.
Project description:Ins(1,4,5)P3 3-kinase is a key enzyme in the regulation of Ins(1,4,5)P3. Overexpression of Ins(1,4,5)P3 3-kinase inhibited agonist-evoked and Ins(1,3,4,5)P4-evoked Ca2+ entry in Xenopus oocytes, but did not inhibit Ca2+ entry evoked by thapsigargin or non-metabolizable Ins(1,4,5)P3 analogues. The data suggest that Ins(1,4,5)P3 alone plays the crucial role in the activation of capacitative Ca2+ entry by emptying intracellular stores.
Project description:The kinetics of Ins(1,4,5)P3 (InsP3)-stimulated Ca2+ release from intracellular stores are unusual in that submaximal concentrations of InsP3 rapidly release only a fraction of the InsP3-sensitive Ca2+ stores. By measuring unidirectional 45Ca2+ efflux from permeabilized rat hepatocytes, we demonstrate that such quantal responses to InsP3 occur at all temperatures between 2 and 37 degrees C, but at much lower rates at the lower temperatures. Preincubation with submaximal concentrations of InsP3, which themselves evoked quantal Ca2+ release, had no effect on the sensitivity of the stores to further additions of InsP3. The final Ca2+ content of the stores was the same whether they were stimulated with two submaximal doses of InsP3 or a single addition of the sum of these doses. Such incremental responses and the persistence of quantal behaviour at 2 degrees C indicate that InsP3-evoked receptor inactivation is unlikely to be the cause of quantal Ca2+ mobilization. Reducing the Ca2+ content of the intracellular stores by up to 45% did not affect their sensitivity to InsP3, but substantially reduced the time taken for each submaximal InsP3 concentration to exert its full effect. These results suggest that neither luminal nor cytosolic Ca2+ regulation of InsP3 receptors are the determinants of quantal behaviour. Our results are not therefore consistent with incremental responses to InsP3 depending on mechanisms involving attenuation of InsP3 receptor function by cytosolic or luminal Ca2+ or by InsP3 binding itself. We conclude that incremental activation of Ca2+ release results from all-or-nothing emptying of stores with heterogeneous sensitivities to InsP3. These characteristics allow rapid graded recruitment of InsP3-sensitive Ca2+ stores as the cytosolic InsP3 concentration increases.
Project description:Ins(1,4,5)P3 is the intracellular messenger that in many cells mediates the effects of Ca2(+)-mobilizing receptors on intracellular Ca2+ stores. An Ins(1,4,5)P3 receptor from cerebellum has been purified and functionally reconstituted, but the relationship between this protein and the high-affinity Ins(1,4,5)P3-binding sites of peripheral tissues is unclear. We compared the Ins(1,4,5)P3-binding sites of liver and cerebellum by measuring inhibition of specific Ins(1,4,[32P]5)P3 binding by various ligands under equilibrium conditions, and find that each ligand binds with similar affinity in the two tissues. Earlier studies in which Ins(1,4,5)P3 binding and Ca2+ mobilization were measured under different conditions demonstrated large differences between KD values for binding and EC50 values (concn. giving half-maximal effect) for Ca2+ release. We show here that, when measured under identical conditions, KD and EC50 values for four agonists are similar. Schild analysis of inhibition of Ins(1,4,5)P3 binding by ATP demonstrates a competitive interaction between the two at the liver Ins(1,4,5)P3-binding site, and this partly accounts for earlier discrepancies in binding and Ca2(+)-release data. We conclude that the high-affinity Ins(1,4,5)P3-binding site of hepatocytes is likely to be the receptor that mediates Ca2+ mobilization, and that this receptor is at present indistinguishable from that in cerebellum.
Project description:In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA)-type Ca(2+)-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca(2+)-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca(2+)-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca(2+)-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.