Regulation of inositol 1,4,5-trisphosphate 3-kinases by calcium and localization in cells

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) 3-kinases (IP(3)Ks) are a group of calmodulin-regulated inositol polyphosphate kinases (IPKs) that convert the second messenger Ins(1,4,5)P(3) into inositol 1,3,4,5-tetrakisphosphate. However, what they contribute to the complexities of Ca(2+) signaling, and how, is still not fully understood. In this study, we have used a simple Ca(2+) imaging assay to compare the abilities of various Ins (1,4,5)P(3)-metabolizing enzymes to regulate a maximal histamine-stimulated Ca(2+) signal in HeLa cells. Using transient transfection, we overexpressed green fluorescent protein-tagged versions of all three mammalian IP(3)K isoforms, including mutants with disrupted cellular localization or calmodulin regulation, and then imaged the Ca(2+) release stimulated by 100 microm histamine. Both localization to the F-actin cytoskeleton and calmodulin regulation enhance the efficiency of mammalian IP(3)Ks to dampen the Ins (1,4,5)P(3)-mediated Ca(2+) signals. We also compared the effects of the these IP(3)Ks with other enzymes that metabolize Ins(1,4,5)P(3), including the Type I Ins(1,4,5)P(3) 5-phosphatase, in both membrane-targeted and soluble forms, the human inositol polyphosphate multikinase, and the two isoforms of IP(3)K found in Drosophila. All reduce the Ca(2+) signal but to varying degrees. We demonstrate that the activity of only one of two IP(3)K isoforms from Drosophila is positively regulated by calmodulin and that neither isoform associates with the cytoskeleton. Together the data suggest that IP(3)Ks evolved to regulate kinetic and spatial aspects of Ins (1,4,5)P(3) signals in increasingly complex ways in vertebrates, consistent with their probable roles in the regulation of higher brain and immune function.     

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.     

Kinetics of intracellular calcium release by inositol 1,4,5-trisphosphate and extracellular ATP in porcine cultured aortic endothelial cells.

Quantitative, time-resolved measurements have been made of intracellular Ca ion release by inositol 1,4,5-trisphosphate (InsP3) and extracellular ATP in porcine aortic endothelial cells in tissue culture. Intracellular free [Ca] was detected with the calcium dye fluo-3 and InsP3 released intracellularly by photolysis of 'caged' InsP3 in whole-cell voltage-clamped aortic endothelial cells. A rise of [Ca] was recorded at InsP3 concentrations greater than 0.2 microM. The timecourse at low InsP3 concentrations comprised a delay of mean 300 ms (range 266-330 ms), a peak in 2-3 s before declining with a half-time of 5-10 s. The delay and time-to-peak decreased with increasing concentrations of InsP3 over the range 0.2-5 microM. At very high concentrations of InsP3 (> 5 microM), the delay in the Ca response was short, always less than 20 ms. The results are consistent with a direct binding and gating action of InsP3 on the Ca channel of the cellular store. Following InsP3 action there is a refractoriness of the InsP3 Ca release process which recovers with a timecourse of half-time about 30 s. A comparison can be made between the timecourse of InsP3 and extracellular ATP actions. High concentrations of ATP (500 microM) acted with a delay of mean 1.8 s (range 1.2-2.5 s), whereas even moderate concentrations of InsP3 acted much more quickly, suggesting that there are slow coupling steps before or during the production of InsP3 in response to extracellular ATP. Both ATP and InsP3 evoked an increase in membrane conductance to K+, probably via Ca.     

Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5-trisphosphate receptor signaling microdomain

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.     

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.     

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.     

Synapse specificity of calcium release probed by chemical two-photon uncaging of inositol 1,4,5-trisphosphate

Biological messengers can be "caged" by adding a single photosensitive group that can be photolyzed by a light flash to achieve spatially and temporally precise biochemical control. Here we report that photolysis of a double-caged form of the second messenger inositol 1,4,5-trisphosphate (IP3) triggers focal calcium release in Purkinje cell somata, dendrites, and spines as measured by two-photon microscopy. In calbindin knock-out Purkinje cells, peak calcium increased with flash energy with higher cooperativity for double-caged IP3 than for conventional single-caged IP3, consistent with a chemical two-photon effect. Spine photolysis of double-caged IP3 led to local calcium release. Uncaging of glycerophosphoryl-myo-inositol 4,5-bisphosphate (gPIP2), a poorly metabolizable IP3 analog, led to less well localized release. Thus, IP3 breakdown is necessary for spine-specificity. IP3- and gPIP2-evoked signals declined from peak with similar, slow time courses, indicating that release lasts hundreds of milliseconds and is terminated not by IP3 degradation but by intrinsic receptor dynamics. Based on measurements of spine-dendrite coupling, IP3-evoked calcium signals are expected to be at least 2.4-fold larger in their spine of origin than in nearby spines, allowing IP3 to act as a synapse-specific second messenger. Unexpectedly, single-caged IP3 led to less release in somata and was ineffective in dendrites and spines. Calcium release using caged gPIP2 was inhibited by the addition of single-caged IP3, suggesting that single-caged IP3 is an antagonist of calcium release. Caging at multiple sites may be an effective general approach to reducing residual receptor interaction.     

Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1.

Our previous studies have demonstrated that calmodulin binds to IP3R type I (IP3R1) in a Ca2+ dependent manner, which suggests that calmodulin regulates the IP3R1 channel. In the present study, we investigated real-time kinetics of interactions between calmodulin and IP3R1 as well as effects of calmodulin on IP3-induced Ca2+ release by purified and reconstituted IP3R1. Kinetic analysis revealed that calmodulin binds to IP3R1 in a Ca2+ dependent manner and that both association and dissociation phase consist of two components with time constants of k(a) = 4.46 x 10(2) and > 10(4) M(-1) s(-1) k(d) = 1.44 x 10(-2) and 1.17 x 10(-1) s(-1). The apparent dissociation constant was calculated to be 27.3 microM. The IP3-induced Ca2+ release through the purified and reconstituted IP3R1 was inhibited by Ca2+/calmodulin, in a dose dependent manner. We interpret our findings to mean that calmodulin binds to IP3R1 in a Ca2+ dependent manner to exert inhibitory effect on IP3R channel activity. This event may be one of the mechanisms governing the negative feedback regulation of IP3-induced Ca2+ release by Ca2+.     

Kinetics of calcium channel opening by inositol 1,4,5-trisphosphate.

The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.     

Temperature and nucleotide dependence of calcium release by myo-inositol 1,4,5-trisphosphate in cultured vascular smooth muscle cells

Inositol 1,4,5-trisphosphate (IP3) rapidly increased 45Ca2+ efflux from a nonmitochondrial organelle in cultured vascular smooth muscle cells that were permeabilized with saponin. A nucleotide, preferably ATP, was essential for IP3-evoked 45Ca2+ release. Two nonhydrolyzable ATP analogues satisfied the nucleotide requirement for IP3-evoked 45Ca2+ release. IP3 strongly stimulated 45Ca2+ efflux at low temperatures (1 to 15 degrees C). Decreasing the temperature from 37 to 4 degrees C inhibited the rate of IP3-stimulated efflux by only about 33%. The failure of such low temperatures to strongly inhibit IP3-induced 45Ca2+ efflux suggests that IP3 activated a Ca2+ channel, rather than a carrier, by a ligand-binding, rather than a metabolic, reaction.     

Effects of inositol 1,4,5-trisphosphate receptor-mediated intracellular stochastic calcium oscillations on activation of glycogen phosphorylase

In various cell types cytosolic calcium (Ca(2+)) is an important regulator. The possible role of Ca(2+) release from the inositol 1,4,5-trisphosphate (IP(3)) receptor channel in the regulation of the phosphorylation-dephosphorylation cycle process involved in glycogen degradation by glycogen phosphorylase have theoretically investigated by using the Li-Rinzel model for cytosolic Ca(2+) oscillations. For the case of deterministic cytosolic Ca(2+) oscillations, there exists an optimal frequency of cytosolic Ca(2+) oscillations at which the average fraction of active glycogen phosphorylase reaches a maximum value, and a mutation for the average fraction of active glycogen phosphorylase occurs at the higher bifurcation point of Ca(2+) oscillations. For the case of stochastic cytosolic Ca(2+) oscillations, the fraction of active phosphorylase is strongly affected by the number of IP(3) receptor channels and the level of IP(3) concentration. Small number of IP(3) receptor channels can potentiate the sensitivity of the activity of glycogen phosphorylase. The average frequency and amplitude of active phosphorylase stochastic oscillations are increased with the level of increasing IP(3) stimuli. The various distributions for the amplitude of active glycogen phosphorylase oscillations in parameters plane are discussed.     

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.     

Effect of inositol 1,4,5-trisphosphate and GTP on calcium release from pituitary microsomes

Microsomal vesicles from bovine anterior pituitary accumulate Ca2+ and maintain a steady-state ambient Ca2+ level of 200 nM. IP3 and GTP both induce calcium release from the microsomal vesicles. The effect of IP3 is inhibited by polyethylene glycol (PEG), and the effect of GTP is absolutely dependent on PEG. Half-maximal effect of IP3 (without PEG) is 0.26 micron, the maximal calcium release attaining 7% of the A23187-releasable pool. The same values for GTP (in the presence of PEG) are 80 microM and 10%, respectively. GTP potentiates the effect of IP3. This potentiation is not mediated by protein phosphorylation.     

Two-state conformational changes in inositol 1,4,5-trisphosphate receptor regulated by calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.     

Kinetics of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate generation in dog-thyroid primary cultured cells stimulated by carbachol

The action of carbachol on the generation of inositol trisphosphate and tetrakisphosphate isomers was investigated in dog-thyroid primary cultured cells radiolabelled with [3H]inositol. The separation of the inositol phosphate isomers was performed by reverse-phase high pressure liquid chromatography. The structure of inositol phosphates co-eluting with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] standards was determined by enzymatic degradation using a purified Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. The data indicate that Ins(1,3,4,5)P4 was the only [3H]inositol phosphate which co-eluted with a [32P]Ins(1,3,4,5)P4 standard, whereas 80% of the [3H]InsP3 co-eluting with an Ins(1,4,5)P3 standard was actually this isomer. In the presence of Li+, carbachol led to rapid increases in [3H]Ins(1,4,5)P4. The level of Ins(1,4,5)P3 reached a peak at 200% of the control after 5-10 s of stimulation and fell to a plateau that remained slightly elevated for 2 min. The level of Ins(1,3,4,5)P4 reached its maximum at 20s. The level of inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] increased continuously for 2 min after the addition of carbachol. Inositol-phosphate generation was also investigated under different pharmacological conditions. Li+ largely increased the level of Ins(1,3,4)P3 but had no effect on Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin, which stimulates dog-thyroid adenylate cyclase and cyclic-AMP accumulation, had no effect on the generation of inositol phosphates. The absence of extracellular Ca2+ largely decreased the level of Ins(1,3,4,5)P4 as expected considering the Ca2(+)-calmodulin sensitivity of the Ins(1,4,5)P3 3-kinase. Staurosporine, an inhibitor of protein kinase C, increased the levels of Ins(1,4,5)P3, Ins(1,3,4,5)P4 and Ins(1,3,4)P3. This supports a negative feedback control of diacyglycerol on Ins(1,4,5)P3 generation.     

The lifetime of inositol 1,4,5-trisphosphate in single cells

In many eukaryotic cell types, receptor activation leads to the formation of inositol 1,4,5-trisphosphate (IP3) which causes calcium ions (Ca) to be released from internal stores. Ca release was observed in response to the muscarinic agonist carbachol by fura-2 imaging of N1E-115 neuroblastoma cells. Ca release followed receptor activation after a latency of 0.4 to 20 s. Latency was not caused by Ca feedback on IP3 receptors, but rather by IP3 accumulation to a threshold for release. The dependence of latency on carbachol dose was fitted to a model in which IP3 synthesis and degradation compete, resulting in gradual accumulation to a threshold level at which Ca release becomes regenerative. This analysis gave degradation rate constants of IP3 in single cells ranging from 0 to 0.284 s-1 (0.058 +/- 0.067 s-1 SD, 53 cells) and a mean IP3 lifetime of 9.2 +/- 2.2 s. IP3 degradation was also measured directly with biochemical methods. This gave a half life of 9 +/- 2 s. The rate of IP3 degradation sets the time frame over which IP3 accumulations are integrated as input signals. IP3 levels are also filtered over time, and on average, large-amplitude oscillations in IP3 in these cells cannot occur with period < 10 s.     

Inositol-1,4,5-trisphosphate releases calcium from skinned cultured smooth muscle cells

We examined the effects of inositol-1,4,5-trisphosphate on 45Ca uptake and 45Ca efflux in the saponin skinned primary cultured rat aortic smooth muscle cells. 10 microM inositol-1,4,5-trisphosphate induced a rapid (half time less than 10 sec) and large quantity of Ca release in both 45Ca uptake and 45Ca efflux in the skinned cells preloaded with 1 microM free Ca. Dose response curves showed that 100 microM inositol-1,4,5-trisphosphate produced a maximal Ca release of 97.3% of the MgATP dependent 45Ca uptake or 289 mumoles/liter cells, which was much greater than the maximal caffeine induced Ca release and would be sufficient to produce maximal tension.     

The effect of inositol 1,4,5-trisphosphate and GTP on calcium release from rat liver microsomes

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and GTP mobilized 8% and 90% of the ionophore-releaseable Ca2+ pool from rat liver microsomes, respectively. In contrast to GTP, which acted after a lag-time, the Ins(1,4,5)P3-induced Ca2+ release was immediate. Poly(ethylene glycol) inhibited the effect of Ins(1,4,5)P3 and enhanced that of GTP. Ins(1,4,5)P3 accelerated and enhanced the GTP-induced Ca2+ release. Guanylyl imidodiphosphate inhibited competitively the GTP stimulated Ca2+ release, but not the GTP-dependent phosphorylation of the Mr 17,000 and 38,000 protein bands.     

Decreased inositol 1,4,5-trisphosphate content in pathogen-challenged soybean cells

Phosphoinositide-specific phospholipase C (PI-PLC) has been shown to be transiently activated when plant cells were treated with elicitors. We thus investigated the activity of PI-PLC when soybean cells were infected with the bacterial pathogen Pseudomonas syringae pv. glycinea, by measuring cellular cytosolic inositol 1,4,5-trisphosphate (IP3) levels. We observed that IP3 content decreased in both compatible and incompatible interactions. In vitro phosphatase activities were similar in both water control and infected cells with slightly lower IP3 degradation observed for infected cells, indicating that the reduced IP3 content in infected cells most likely results from reduced PI-PLC activity. We hypothesize that reduced IP3 content following infection may lead to suppression of various housekeeping activities of the cells, thus diverting the cellular resources either to the synthesis of defense-related compounds against pathogens, and/or to the growth of pathogens.     

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.