Metabolic and functional consequences of introducing inositol 1,4,5-trisphosphate into saponin-permeabilized human platelets.

In an earlier study we reported the effect of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in releasing Ca2+ from highly purified human platelet intracellular membrane vesicles. [Authi & Crawford (1985) Biochem. J. 230, 247-253]. We have now investigated the metabolic and functional consequences of introducing Ins(1,4,5)P3 into saponin-permeabilized platelets. Washed human platelets when resuspended in a suitable medium were permeabilized with saponin (10-14 micrograms/ml) to allow entry of low-Mr water-soluble molecules without significant release of the cytoplasmic marker enzyme protein lactate dehydrogenase. Saponin-permeabilized platelets show identical platelet responses (shape change, aggregation and release of 5-hydroxy[14C]tryptamine) to both collagen (5 micrograms/ml) and thrombin (0.1 unit/ml) as obtained with intact cells, indicating that there is minimal disturbance to the surface membrane receptor topography for these two agonists. Ins(1,4,5)P3 (1-10 microM) added to saponin-treated platelets (but not to intact platelets) induced dose-related shape change, aggregation and release of 5-hydroxy[14C]tryptamine which at maximal doses was comparable with responses obtained with thrombin or collagen. The cyclo-oxygenase inhibitors indomethacin and aspirin, if added prior to saponization and Ins(1,4,5)P3 addition, completely inhibited both aggregation and release of 5-hydroxy[14C]tryptamine (EC50 for indomethacin, 50 nM; for aspirin, 30 microM). We believe that Ins(1,4,5)P3 induces the release of Ca2+ from intracellular storages sites which stimulates the Ca2+-dependent phospholipase A2 releasing arachidonic acid from membrane phospholipids. Arachidonic acid is then converted to the aggregatory prostanoids (prostaglandin H2 and thromboxane A2) resulting in the observed responses. This concept is supported by the use of the thromboxane receptor antagonists EPO 45 and EPO 92, both of which also completely inhibit Ins(1,4,5)P3-induced responses in saponin-permeabilized platelets. Electron microscopy of the platelet preparations revealed that thrombin- and collagen-induced platelet aggregates of intact and saponized cells were identical, showing extensive pseudopod formation and dense granule release. The Ins(1,4,5)P3-induced aggregates also showed similar dense granule release but an almost total absence of pseudopod formation. These results are discussed in the light of the second messenger role of Ins(1,4,5)P3 in stimulus-response coupling in platelets.     

Inositol 1,4,5-trisphosphate induces calcium release from a non-mitochondrial pool in amoebae of Dictyostelium

Evidence is presented for two distinct Ca²⁺ pools in amoebae of Dictyostelium discoideum. One pool, presumably mitochondrial, was sensitive to the mitochondrial inhibitors oligomycin and dinitrophenol and showed an affinity for Ca²⁺ in the μM concentration range. The other Ca²⁺ pool, which was insensitive to these inhibitors, was of lower capacity but had higher affinity (in the nM range). Inositol 1,4,5-trisphosphate (5 μM) added to saponin-permeabilized amoebae induced a rapid release of Ca²⁺ from the latter pool but had no effect on the presumed mitochondrial pool. Controls using addition of inositol 1,4-bisphosphate (the hydrolytic product of IP₃) induced no such Ca²⁺ release. The results provide strong support for the involvement of IP₃ in signal transmission during chemotaxis of D. discoideum.     

Inositol 1,4,5-trisphosphate and 5'-GTP induce calcium release from different intracellular pools

It has been shown recently by several groups that 5'-GTP can release calcium from intracellular compartments independently from inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by a mechanism which seems to be different from that used by Ins(1,4,5)P3. We report here for the first time that the 5'-GTP-sensitive and the Ins(1,4,5)P3-sensitive calcium pools reside in different intracellular compartments.     

Inositol 1,4,5-trisphosphate induced calcium release from corn coleoptile microsomes

The effect of inositol 1,4,5-trisphosphate (IP3) on Ca2+ release from microsomes of corn coleoptiles was investigated. Addition of micromolar concentrations of IP3 to Ca2+ loaded microsomes resulted in rapid release of 20-30% of sequestered Ca2+. Maximal and half maximal Ca2+ release occurred at 20 and 8 microM of IP3 respectively. Part of the Ca2+ released by IP3 was reaccumulated into microsomes within 4 min. The amount of Ca2+ released by IP3 was found to be dependent on free Ca2+ concentration in the incubation medium at the time of release. Maximum Ca2+ release was observed around 0.1 microM free Ca2+ concentration in the assay medium. These data suggest that IP3 might act as a second messenger in plants in a manner similar to animal systems by altering cytosolic levels of calcium.     

Inositol 1,4,5-trisphosphate induces cyclic GMP formation in Dictyostelium discoideum

Chemotactic signalling in the cellular slime mould Dictyostelium discoideum employs signalling molecules such as folate and cyclic AMP. These bind to specific cell surface receptors and rapidly trigger internal responses that induce chemotactic movement of the amoebae. Previous studies have shown that actin is polymerised within 3-5 sec of cyclic AMP or folate binding and that a peak of cyclic GMP is formed within 9-12 sec. Release of Ca2+ from intracellular stores has been implicated as a secondary messenger. Here we present evidence that D-myo-inositol 1,4,5-trisphosphate, when added to permeabilized amoebae of Dictyostelium, can mimic the action of chemoattractants on normal intact amoebae in inducing cyclic GMP formation. Our data suggest that IP3, which is known to act as an intermediary messenger between cell surface hormone receptors and release of Ca2+ from internal stores in mammalian cells, functions in a similar capacity during chemotaxis of this primitive eukaryote.     

Inositol 1,4,5-trisphosphate releases Ca2+ from a Ca2+-transporting membrane vesicle fraction derived from human platelets

Human platelet membrane vesicles that accumulated Ca2+ in the presence of ATP were isolated on an isoosmotic KCl-Percoll gradient. ATP-dependent Ca2+ uptake was stimulated by oxalate and phosphate to steady-state levels of greater than 100 nmol/mg protein, and the accumulated Ca2+ could be largely released by ionophore A23187. Inositol 1,4,5-trisphosphate, in a dose-dependent manner (0.5-5.0 microM), caused the rapid release (less than 5 s) of 40-70% of the total A23187-releasable store of accumulated Ca2+. The membrane vesicles that release accumulated Ca2+ in response to inositol 1,4,5-trisphosphate were enriched in enzymes characteristically found in smooth endoplasmic reticulum. These results support the hypothesis that inositol 1,4,5-trisphosphate, produced by the hydrolysis of phosphatidylinositol 1,4-bisphosphate in response to stimulation of cell surface receptors, is a second messenger mediating the release of Ca2+ from intracellular storage sites.     

Inositol 1,4,5-trisphosphate mobilizes glucose-incorporated calcium from pancreatic islets

Mobilization of intracellular calcium from beta-cell-rich pancreatic islets of ob/ob-mice was studied by measuring unidirectional 45Ca efflux at 37 degrees and 18 degrees C during perifusion with a K+-rich medium deficient in Ca2+ and Na+. Addition of 100 microM carbachol induced a prominent peak of Ca2+ efflux from islets preexposed to glucose. After cell permeabilization with digitonin D-myo-inositol 1,4,5-trisphosphate (IP3) caused glucose-dependent mobilization of calcium. In demonstrating that not only carbachol but also IP3 can mobilize calcium incorporated in response to glucose, the present data suggests that the endoplasmic reticulum participates in glucose-induced lowering of cytoplasmic Ca2+ activity in the pancreatic beta-cells.     

Inositol 1,4,5-trisphosphate and guanine nucleotides activate calcium release from endoplasmic reticulum via distinct mechanisms

A sensitive and specific guanine nucleotide regulatory process has recently been shown to rapidly mediate a substantial release of Ca2+ from endoplasmic reticulum within the N1E-115 neuronal cell line (Gill, D. L., Ueda, T., Chueh, S. H., and Noel, M. W. (1986) Nature 320, 461-464). The relationship between this mechanism and Ca2+ efflux mediated by the intracellular regulator inositol 1,4,5-trisphosphate (IP3) has been investigated. Using saponin-permeabilized N1E-115 cells, studies reveal a number of distinctions between the activation of Ca2+ release mediated by GTP and IP3. Thus, the GTP-mediated Ca2+ release process is specifically activated by polyethylene glycol which increases both GTP sensitivity and the extent of GTP-activated Ca2+ release; in contrast, IP3-dependent Ca2+ release is unaffected by polyethylene glycol. The non-hydrolyzable GTP analogue guanosine 5'-O-(3-thio)triphosphate, which completely inhibits GTP-mediated Ca2+ release, does not alter release mediated by IP3. Decreasing the release temperature from 37 to 4 degrees C decreases IP3-activated Ca2+ release by only 20%, whereas the action of GTP on Ca2+ release is abolished at 4 degrees C. Activation of Ca2+ release by IP3 is completely inhibited by increasing free Ca2+ from 0.1 to 10 microM, whereas the fraction of GTP-dependent Ca2+ release (approximately 50% of ionophore-releasable Ca2+) remains unaltered with increasing free Ca2+. These distinctions between IP3- and GTP-mediated Ca2+ release indicate that the two effectors function via distinct mechanisms to activate Ca2+ release; however, they do not preclude the possibility that coupling between the two mechanisms can occur or that a common Ca2+-translocating pathway activated by both effectors exists.     

Inositol 1,4,5-trisphosphate receptor isoforms show similar Ca2+ release kinetics

The inositol 1,4,5-trisphosphate receptor (InsP3R) is an intracellular Ca2+ release channel which upon activation initiates many cellular functions. Multiple InsP3R subtypes are expressed in most cell types but the physiological significance of this heterogeneity is poorly understood. This study has directly compared the functional properties of the three different InsP3R isoforms by analyzing their InsP3-induced Ca2+ release (IICR) properties in cell lines which predominantly express each isoform subtype. The InsP3-dependence of the amount or extent of IICR was InsP3R isoform-specific, with the type III isoform having the lowest affinity with respect to Ca2+ release. The transient kinetics of IICR, measured using stopped-flow spectrofluorimetry, however, were similar for all three InsP3R isoforms. At maximal InsP3 concentrations (20 microM) the rate constants where between 0.8 and 1.0 s(-1) for the fast phase and 0.25-0.45 s(-1) for the slow phase. The concentration of InsP3 required to induce half-maximal rates of Ca2+ release (EC50) were also similar for the three isoforms (0.2-0.4 microM for the fast phase and 0.75-0.95 microM for the slow phase). These results indicate the InsP3R channel does not significantly differ functionally in terms of Ca2+ release rates between isoforms. The temporal and spatial features of intracellular Ca2+ signals are thus probably achieved through InsP3R isoform-specific regulation or localization rather than their intrinsic Ca2+ efflux properties.     

Isolation of a phosphomonoesterase from human platelets that specifically hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate

Platelets, and a variety of other cells, rapidly hydrolyze the phosphoinositides in response to stimulation by agonists. One of the products of hydrolysis of phosphatidylinositol 4,5-diphosphate is inositol 1,4,5-trisphosphate, which recently has been suggested to mediate intracellular Ca2+ mobilization. We have found that human platelets contain an enzyme that degrades inositol 1,4,5-trisphosphate. We have isolated this soluble enzyme and find that it hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate (Km = 30 microM, Vmax = 5.3 microM/min/mg of protein). The products of the reaction are inositol 1,4-diphosphate and phosphate. The apparent molecular weight of the enzyme is 38,000 as determined both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol. This enzyme is specific for inositol 1,4,5-trisphosphate. Other water soluble inositol phosphates as well as phosphorylated sugars are not hydrolyzed, while the only inositol containing phospholipid hydrolyzed is phosphatidylinositol 4,5-diphosphate at a rate less than 1% that for inositol 4,5-trisphosphate. The inositol 1,4,5-trisphosphate 5-phosphomonoesterase requires Mg2+ for activity and is inhibited by Ca2+, Ki = 70 microM. Li+, up to 40 mM, has no effect on enzyme activity. The duration and magnitude of any inositol 1,4,5-trisphosphate response in stimulated platelets may be determined by the activity of this enzyme.     

Formation and metabolism of inositol 1,4,5-trisphosphate in human platelets.

1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.     

Inhibitory action of cyclic AMP on inositol 1,4,5-trisphosphate-induced Ca2+ release in saponin-permeabilized platelets

Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) has been measured in saponin-permeabilized human platelets with quin2 or 45Ca2+. Ca2+ was sequestered by intracellular organelles in the presence of ATP, and IP3 released half of the sequestered Ca2+. The addition of cyclic AMP (cAMP) to permeabilized platelets transiently accelerated Ca2+ sequestration, but did not alter the steady-state level. In contrast, IP3-induced Ca2+ release was greatly inhibited by cAMP. Phorbol myristate acetate, an activator of protein kinase C did not affect IP3-induced Ca2+ release. These results indicate that cAMP may be involved in the regulation of IP3-induced Ca2+ release in human platelets.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Inositol 1,4,5-triphosphate induces calcium release from a non- mitochondrial pool in amoebae of Dictyostelium

Evidence is presented for two distinct CA2+ pools in amoebae of Dictyostelium discoideum. One pool, presumably mitochondrial, was sensitive to the mitochondrial inhibitors oligomycin and dinitrophenol and showed an affinity for Ca2+ in the micro M concentration range. The other Ca2+ pool, which was insensitive to these inhibitors, was of lower capacity but had higher affinity (in the nM range). Inositol 1,4,5-trisphosphate (5 micro M) added to saponin-permeabilized amoebae induced a rapid release of Ca2+ from the latter pool but had no effect on the presumed mitochondrial pool. Controls using addition of inositol 1,4-bisphosphate (the hydrolytic product of IP3) induced no such CA2+ release. The results provide strong support for the involvement of IP3 in signal transmission during chemotaxis of D. discoideum.     

Inositol 1,4,5-trisphosphate induced mobilization of Ca2+ from rat brain synaptosomes

Inositol 1,4,5-trisphosphate (IP₃) was found to release Ca²⁺ from presynaptic nerve endings (synaptosomes) made permeable with saponin. ATP-dependent Ca²⁺ uptake was carried out until equilibrium was reached. Addition of IP₃ produced a rapid release of Ca²⁺, which was complete within 60 sec, followed by Ca²⁺ reaccumulation to the original level in 5–7 min. Cholinergic receptor stimulation with muscarine also produced a similar Ca²⁺ release from synaptic endoplasmic reticulum. Ca²⁺ release by IP₃ was not detectable in the absence of the mitochondrial inhibitors oligomycin or sodium azide. Reaccumulation of Ca²⁺ was prevented by the presence of vanadate, a potent inhibitor of Ca²⁺/Mg²⁺ ATPase. Half maximal and near complete release of Ca²⁺ took place at 0.4 M and 3 M IP₃ concentrations, respectively. These studies demonstrate for the first time IP₃ mobilization of Ca²⁺ from endoplasmic reticulum within synaptic plasma membranes.     

Inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate formation in Ca2+-mobilizing-hormone-activated cells.

The inositol trisphosphate liberated on stimulation of guinea-pig hepatocytes, pancreatic acinar cells and dimethyl sulphoxide-differentiated human myelomonocytic HL-60 leukaemia cells is composed of two isomers, the 1,4,5-trisphosphate and the 1,3,4-trisphosphate. Inositol 1,4,5-trisphosphate was released rapidly, with no measurable latency on hormone stimulation, and, consistent with its proposed role as an intracellular messenger for Ca2+ mobilization, there was good temporal correlation between its formation and Ca2+-mediated events in these tissues. There was a definite latency before an increase in the formation of inositol 1,3,4-trisphosphate could be detected. In all of these tissues, however, it formed a substantial proportion of the total inositol trisphosphate by 1 min of stimulation. In guinea-pig hepatocytes, where inositol trisphosphate increases for at least 30 min after hormone application, inositol 1,3,4-trisphosphate made up about 90% of the total inositol trisphosphate by 5-10 min. In pancreatic acinar cells, pretreatment with 20 mM-Li+ caused an increase in hormone-induced inositol trisphosphate accumulation. This increase was accounted for by a rise in inositol 1,3,4-trisphosphate; inositol 1,4,5-trisphosphate was unaffected. This finding is consistent with the observation that Li+ has no effect on Ca2+-mediated responses in these cells. The role, if any, of inositol 1,3,4-trisphosphate in cellular function is unknown.     

Inositol 1,4,5-trisphosphate mobilizes intracellular Ca2+ from permeabilized insulin-secreting cells.

A possible role in secretory processes is proposed for inositol 1,4,5-triphosphate (IP3), based upon investigations of the Ca2+ steady state maintained by "leaky', insulin-secreting RINm5F cells. These cells had been treated with digitonin to permeabilize their plasma membranes and thereby ensure that only intracellular Ca2+ buffering mechanisms were active. When placed in a medium with a cation composition resembling that of the cytosol, cells rapidly took up Ca2+ as measured by a Ca2+-specific minielectrode. Two Ca2+ steady states were observed. A lower level of around 120nM required ATP-dependent Ca2+ uptake and was probably determined by the endoplasmic reticulum. The higher steady state (approx. 800 nM), seen only in the absence of ATP, was shown to be due to mitochondrial activity. IP3 specifically released Ca2+ accumulated in the ATP-dependent pool, but not from mitochondria, since Ca2+ release was demonstrated in the presence of the respiratory poison antimycin. The IP3-induced Ca2+ release was rapid, with 50% of the response being seen within 15s. The apparent Km was 0.5 microM and maximal concentrations of IP3 (2.5 microM) produced a peak Ca2+ release of 10 nmol/mg of cell protein, which was followed by re-uptake. A full Ca2+ response was seen if sequential pulses of 2.5 microM-IP3 were added at 20 min intervals, although there was a slight (less than 20%) attenuation if the intervening period was decreased to 10 min. These observations could be related to the rate of IP3 degradation which, in this system, corresponded to a 25% loss of added 32P label within 2 min, and a 75% loss within 20 min. The results suggest that IP3 might act as a link between metabolic, cationic and secretory events during the stimulation of insulin release.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.