Messenger IP3 Research Articles

Frying Up a New Stress Regulator

Using a screen to identify Arabidopsis mutants that have abnormal responses to abscisic acid (ABA) or stress conditions, Xiong et al. identified a mutant, called fiery (fry), that has higher-than-normal, nonadaptive responses to stressful stimuli. Under cold temperature conditions, high salt, or ABA treatment, fry1 plants harboring a combination stress-responsive and ABA-responsive reporter gene exhibited greater-than-normal levels of reporter gene expression. Even at temperatures slightly below that of normal room temperature, expression from the gene reporter in fry1 plants was greater than normal, suggesting that fry1 reduced the threshold of stress-dependent gene expression. fry1 plants were defective for stress tolerance as measured by ion leakage under cold or drought-mimicking conditions, and increased plant death was exhibited under high salt concentrations. The fry1 mutation was mapped and the gene isolated, the sequence of which predicted an inositol polyphosphate 1-phosphatase. Chimeric GST-Fry1 (wild-type) proteins dephosphorylated substrates in vitro, whereas mutant Fry1 chimeric proteins were devoid of catalytic activity. Under stress conditions, fry1 plants produced and accumulated large amounts of inositol trisphosphate (IP3), suggesting that impaired turnover of the second messenger IP3 may result in hypersensitivity to stress and ABA stimuli.L. Xiong, B.-h. Lee, M. Ishitani, H. Lee, C. Zhang, J.-K. Zhu, FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev. 15, 1971-1984 (2001). [Abstract] [Full Text]

Participation of the phosphoinositide metabolism in the hypersensitive response of Citrus limon against Alternaria alternata.

Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. 3.1.4.3) inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata.

Cyclic Peptides as Molecular Adapters for a Pore-Forming Protein

Recently, cyclodextrins were shown to act as adapters for the pore-forming protein staphylococcal α-hemolysin (αHL). The bagel-shaped molecules bind in the lumen of the transmembrane channel formed by αHL and alter the properties of the pore. For example, the unitary conductance is reduced, and organic molecules can act as channel blockers by binding to the cavity within the cyclodextrin adapter. In a search for a class of adapters more versatile than the cyclodextrins and amenable to preparation as libraries, cyclic peptides have now been examined. Four peptides, cyclo[(l-Arg-d-Leu)4-] ((RL)4), cyclo[(l-Glu-d-Leu)4-] ((EL)4), cyclo[(-l-Phe-d-N-(aminoethyl)-Ala-l-Phe-d-Ala)2-] (diNH3+-(FA)4), and cyclo[(-l-Phe-d-N-(carboxymethyl)-Ala-l-Phe-d-Ala)2-] (diCO2--(FA)4), became lodged within the αHL pore, altering the unitary conductance and ion selectivity. Further, the positively charged peptides, (RL)4 and diNH3+-(FA)4, were able to act as binding sites for various small polyanions. For example, the second messenger IP3 bound to (RL)4 within the αHL pore. Therefore, the modulation of single-channel currents through pores containing cyclic peptide adapters may prove useful for sensing a variety of molecules.

5‐HT2A receptors and their second messenger IP3 are only correlated in human brain and not in platelets

5-HT2A receptors and their second messenger IP3 are only correlated in human brain and not in platelets.

5-HT2A receptors and their second messenger IP3 are only correlated in human brain and not in platelets

5-HT2A receptors and their second messenger IP3 are only correlated in human brain and not in platelets

5‐HT2A receptors and their second messenger IP3 are only correlated in human brain and not in platelets

The 5-HT2A postsynaptic receptor and its second messenger inositol 1,4,5-trisphosphate (IP3) concentrations were simultaneously determined in frontal cortex from 17 control brains and in platelets from 43 control subjects. IP3 values obtained in human frontal cortex were 3.08 ± 0.63 pmol/gr of neural tissue. The maximum binding of 5-HT2A receptors in this brain region was 551 ± 42 fmol/mg, with mean Kd values of 1.3 ± 0.07 nM. A significant positive correlation was observed between the 5-HT2A binding density and its second messenger IP3 concentrations (r=0.54; p=0.025). In human platelet, a mean IP3 value of 0.151 ± 0.01 pmol/109 platelets was observed, with 5-HT2A Bmax and Kd values of 119 ± 5 fmol/mg and 1.24 ± 0.04 nM respectively. No significant correlation was observed between the 5-HT2A binding density and the IP3 concentrations (r=0.01; p=0.91). These results confirm that although IP3 is the second messenger related to the 5-HT2A receptor activation both in human brain and in platelets, the human platelet seems to possess other regulation mechanisms related to second messenger activation.

Platelet serotonin S-HT 2A binding sites and second messenger IP 3 in schizophrenia

Platelet serotonin S-HT 2A binding sites and second messenger IP 3 in schizophrenia

Different regional distribution of the 5-HT reuptake complex, the 5-HT2A receptors and their second messenger IP3 in human brain

The serotonin reuptake complex labelled with [3H]imipramine and [3H]paroxetine, the 5-HT2A postsynaptic receptor labelled with [3H]ketanserin and the inositol trisphosphate (IP3) concentrations were simultaneously determined in several human brain regions. An inverse distribution was found between the pre and postsynaptic receptors. Thus, while the serotonin reuptake complex labelled with either [3H]imipramine and [3H]paroxetine displayed a maximum density in subcortical areas and basal ganglia, the 5-HT2A postsynaptic receptor labelled with [3H]ketanserin showed higher binding values in cortical areas. The apparent affinity constants for the three radioligands were similar over the 12 regions studied (approx 1nM for [3H] imipramine, approx 0.06 nM for [3H]paroxetine and approx 1nM for [3H]ketanserin). IP3 levels were highest in frontal cortex (6.3 ± 2.4 pmol/gr) followed by substantia nigra (4.6 ± 1.5 pmol/gr). The remaining regions exhibited approximately the same concentration range (two or three fold lower than frontal cortex values). Frontal cortex was the only brain region showing a significant correlatioin between the 5-HT2A maximum binding and the levels of its second messenger IP3.

Different regional distribution of the 5‐HT reuptake complex, the 5‐HT2A receptors and their second messenger IP3 in human brain

The serotonin reuptake complex labelled with [3H]imipramine and [3H]paroxetine, the 5-HT2A postsynaptic receptor labelled with [3H]ketanserin and the inositol trisphosphate (IP3) concentrations were simultaneously determined in several human brain regions. An inverse distribution was found between the pre and postsynaptic receptors. Thus, while the serotonin reuptake complex labelled with either [3H]imipramine and [3H]paroxetine displayed a maximum density in subcortical areas and basal ganglia, the 5-HT2A postsynaptic receptor labelled with [3H]ketanserin showed higher binding values in cortical areas. The apparent affinity constants for the three radioligands were similar over the 12 regions studied (approx 1nM for [3H] imipramine, approx 0.06 nM for [3H]paroxetine and approx 1nM for [3H]ketanserin). IP3 levels were highest in frontal cortex (6.3 ± 2.4 pmol/gr) followed by substantia nigra (4.6 ± 1.5 pmol/gr). The remaining regions exhibited approximately the same concentration range (two or three fold lower than frontal cortex values). Frontal cortex was the only brain region showing a significant correlatioin between the 5-HT2A maximum binding and the levels of its second messenger IP3.

Function and molecular basis of action of vasopressin 4—8 and its analogues in rat brain

VP 4-8 as a highly potent behavioral-active metabolite of arginine-vasopressin (VP) has been studied in detail at four levels, i.e. ligand level, membrane binding level, intracellular level and nuclear level. The purpose of this chapter is to review and discuss the main results obtained from our recent pharmacological and biochemical investigations which are described as follows: 1, structure-function relationship of VP 4-8 and its analogs; 2, some characters of VP 4-8-specific binding, the distribution of the binding sites in the rat brain and the consequent effect on long-term potentiation of synaptic transmission; 3, a putative receptor-mediated signaling pathway involving second messenger IP3, immediately-early gene c-fos transcription and protein kinase PKC, CaMKII and MAPK; 4, peptide-induced enhancement of some crucial functional proteins such as calmodulin, nerve growth factor (NGF) and brain-derived nerve growth factor (BDNF). The physiological significance of the events following VP 4-8 administration and particularly, its possible role in learning and memory processes are discussed.

Volatile anaesthetics have differential effects on recombinant m1 and m3 muscarinic acetylcholine receptor function

Muscarinic acetylcholine signalling plays major roles in regulation of consciousness, cognitive functioning, pain perception and circulatory homeostasis. Halothane has been shown to inhibit m1 muscarinic signalling. However, no comparative data are available for desflurane, sevoflurane or isoflurane, nor have the anaesthetic effects on the m3 subtype (which is also prominent in the brain) been studied. Therefore, we have investigated the effects of these compounds on isolated m1 and m3 muscarinic receptor function. Defolliculated Xenopus oocytes expressing recombinant m1 or m3 muscarinic or (for comparison) AT1A angiotensin II receptors were voltage clamped, and Ca(2+)-activated Cl- currents (ICl(Ca)) induced by acetyl-beta-methylcholine (Mch) or angiotensin II were measured in the presence of clinically relevant concentrations of halothane, sevoflurane, desflurane or isoflurane. To determine the site of action of the volatile anaesthetics we compared anaesthetic effects on m1, m3 and AT1A receptor function and studied the effects of volatile anaesthetics on signalling induced by intracellular injection of the second messenger IP3. Desflurane had a biphasic effect on m1 signalling, enhancing at a concentration of 0.46 mmol litre-1 but depressing at 0.92 mmol litre-1. A similar, although not significant, trend was observed with m3 signalling. Isoflurane had no effect on m1 signalling, but profoundly inhibited m3 signalling. Sevoflurane depressed the function of m1 and m3 signalling in a dose-dependent manner. Halothane, similar to its known effect on m1 signalling, dose-dependently depressed m3 function. ICl(Ca) induced by intracellular injections of IP3 were unaffected by all four anaesthetics. Similarly, none of the anaesthetics tested affected AT1A signalling. Absence of interference with AT1A signalling and intracellular pathways suggest that the effects of anaesthetics on muscarinic signalling most likely result from interactions with the m1 or m3 receptor molecule. Multiple interaction sites with different affinities may explain the biphasic response to desflurane. Anaesthetic-specific effects on closely related receptor subtypes suggest defined sites of action for volatile anaesthetics on the receptor protein.

Impaired contractile G protein function in antrum and colon circular muscle from pregnant guinea pigs

It has been previously shown that pregnancy has an inhibitory effect on the motility of gastrointestinal (GI) tract. We therefore examined the mechanisms responsible for this impaired GI motility in pregnant guinea pigs. Antrum and left colon were removed from adult third-trimester pregnant guinea pigs and nonpregnant female guinea pigs (control). Circular smooth muscle ceils were isolated by enzymatic digestion with collagenase and permeabilized by brief exposure to saponin as needed. Cell contraction was expressed as percent shortening with respect to control muscle cells. Contraction in response to CCK-8 (0.1 pM to 0.1 laM) was significantly reduced in both antrum and colon circular muscle from pregnant guinea pigs with a maximal shortening of only 14.2-+0.2% and 14.5 -+0.7% respectively when compared to 22.5 -+ 1.1% and 21.7 -+0.9% shortening in control animals (p<0.01, by ANOVA). Contraction in response to direct G protein activation with GTP,/S was also significantly reduced in antrum and colon circular muscle cells from pregnant guinea pigs. GTP)'S at 10 laM caused a maximal shortening of only 11.9 -+ 0.8% and 11.3 -+ 0.7% in antrum and colon circular muscle ceils from pregnant animals, respectively, as opposed to 20.1:1: 0.4% and 20.5 -+ 1.1% in controls. In contrast, the contraction of both antrum and colon circular muscle cells induced by a second messenger IP3 was similar between pregnant and control guinea pigs. Furthermore, the contractile responses to potassium chloride, which is a receptor-G protein independent agonist, were not significantly different from controls, These data suggested that pregnancy might affect the function of G proteins in the GI circular smooth muscle. To further confirm this hypothesis, the function of G proteins in antrum and colon circular muscle from both groups of animals was assessed by using [35S]GTP'¢S binding. Crude membranes derived from antrum and colon circular muscle were incubated with 30 nM [35S]GTPTS with or without agonist stimulation. CCK-8 at 1paM caused a significant increase of [35S]GTP)'S binding to Gq/1 let over the basal level (without CCK), but not to Gietl-2, Giet3, or Gsa. The stimulation of Gq/1 let binding was significantly reduced in antrum and colon circular muscle from pregnant guinea pig when compared to that from control animals (p<0.05, by t test). However, VIP at 1 taM caused a significant increase of [35S]GTPyS binding to Gset, but not to Gq/llet, Gietl-2 or Giot3. The stimulation of Gset binding induced by VIP was similar between these two groups. In conclusion, pregnancy appears to impair gastrointestinal muscle contractility by downregulating certain G proteins that mediate muscle contraction such as Gq/1 let protein. This research was supported by NIH grant R01-DK27389.

Augmentation of lithium's behavioral effect by inositol uptake inhibitors.

Lithium inhibits the enzyme inositol monophosphatase and thus obstructs the enzymatic degradation of inositol triphosphate (IP3) to inositol in the phosphate-phosphoinositide (PIP) cycle. This inhibition may result in reduced availability of the second messengers IP3 and DAG that are derivates of the PIP cycle, and this action is currently a leading hypothesis regarding lithium's therapeutic and prophylactic effect in affective disorders. Inositol is also available to the cell by uptake from the intercellular matrix, and therefore it is possible that compounds that block the uptake may have lithium-like effects. To test this hypothesis, the present study evaluates the effects of two inositol uptake inhibitors, the carbohydrate L-fucose and the cyclodepsipeptide nordidemnin, in a behavioral model of pilocarpine-induced seizures known to be enhanced by lithium. We tested the possibility that L-fucose produces lithium-like effects, or that L-fucose or nordidemnin augment lithium's behavioral effects. Results indicate that acute ICV treatment with L-fucose did not by itself have a lithium-like effect in the behavioral model, but significantly augmented lithium's effect when combined with lithium treatment. Nordidemnin treatment showed similar effects. The results suggest that when inositol monophosphatase is inhibited by lithium, further restriction of cellular inositol availability may result in an augmentation of lithium's behavioral effects. It is possible that such manipulations may be applicable in the treatment of patients with affective disorders, especially patients who are poor responders to lithium monotherapy.

Swelling and damage of glial cells by lactacidosis and glutamate: effect of α-trinositol

The therapeutical efficacy of α-trinositol ( d- myo-inositol-1,2,6-trisphosphate), an isomer of the intracellular messenger IP 3, was analyzed on cytotoxic swelling and damage of glial cells in vitro from lactacidosis or glutamate. C6 glioma cells suspended in a physiological medium were either exposed to pH 5.0 by administration of lactic acid, or to 1 mM glutamate. Cell swelling and viability were quantified by flow cytometry. Lactacidosis of pH 5.0 led to an increase in cell volume to 139.7±1.3% within 20 min whereas α-trinositol was reducing the swelling response by approximately 25% ( P<0.01). In addition, at pH 5.0 the fraction of viable cells was lowered from 94.3±0.2% (control) to only 53.8±3.1% after 60 min. α-Trinositol was found to protect also cell viability; at 60 min of lactacidosis 70.2±1.6% of the cells still were viable ( P<0.01). The addition of glutamate (1 mM) to the cell suspension led to a steady increase in cell size, reaching 110% of control at 120 min, irrespectively of whether α-trinositol was added or not.

Salutary effects of ATP-MgCl2 on altered hepatocyte signal transduction after hemorrhagic shock.

Although previous studies indicate that hepatocyte P2 purinoceptors, macrophage adenosine 3',5'-cyclic monophosphate (cAMP), and beta-adrenergic receptors decrease after hemorrhage and that administration of ATP-MgCl2 after hemorrhage normalizes these parameters, it is not known whether other aspects of hepatocyte signal transduction processes, such as transmembrane coupling, are also affected by hemorrhage and, if so, whether ATP-MgCl2 has any beneficial effects on signal transduction. To study this, rats underwent a 5-cm midline laparotomy (i.e., trauma induced) and were bled to and maintained at a mean arterial pressure of 40 mmHg until 40% of a maximum bleed-out volume was returned in the form of Ringer lactate (RL). They were then resuscitated with three times the volume of shed blood with RL over 45 min followed by two times the volume with RL+ATP-MgCl2 (50 mumol/kg body wt) or an equivalent volume of saline over 95 min. Hepatocytes were isolated at 4 and 27 h after resuscitation, and basal as well as stimulated levels of cAMP and inositol 1,4,5-trisphosphate (IP3) were determined. The results indicate that basal levels of cAMP decreased whereas IP3 increased after hemorrhage and resuscitation. Receptor-dependent stimuli (i.e., glucagon and vasopressin) failed to elicit cAMP or IP3 accumulation after hemorrhage. In contrast, receptor-independent stimulation was not impaired. ATP-MgCl2 treatment, however, prevented the decreased basal levels of cAMP and IP3 and the ability of hepatocytes to respond to receptor-dependent stimulation. Thus ATP-MgCl2 treatment of animals after trauma-hemorrhage and resuscitation attenuates the impaired second messengers cAMP and IP3 and their membrane transduction processes.

Genetics of phototaxis in a model eukaryote, Dictyostelium discoideum.

The life cycle of Dictyostelium discoideum offers a unique opportunity to study signal transduction in eukaryotic cells at both the unicellular and multicellular levels of organization. Adding to the already extensive knowledge of the unicellular stages, classical and molecular genetics have begun to unravel transduction of signals controlling morphogenesis and behaviour (phototaxis and thermotaxis) in the multicellular 'slug' stage of the life cycle. Distributed over all seven genetic linkage groups are probably about 20, but possibly as many as 55, genes of importance for slug behaviour. The encoded proteins appear from pharmacological studies and mutant phenotypes to govern transduction pathways involving the intracellular second messengers cyclic AMP, cyclic GMP, IP3 and Ca2+. Pathways from the photo- and thermoreceptors converge first with each other and thence, at the level of the second messengers, with those from extracellular tip activation (cyclic AMP) and inhibition (Slug Turning Factor and/or ammonia and/or adenosine) signals that control slug movement and morphogenesis.

Intracellular calcium-release channels: regulators of cell life and death

Intracellular Ca2+-release channels on the sarcoplasmic reticulum of striated muscle [ryanodine receptors (RyRs)] and on the endoplasmic reticulum of almost all types of cells [inositol 1,4,5-trisphosphate receptors (IP3Rs)] comprise a unique family of molecules that are structurally and functionally distinct from all other known ion channels. These channels play crucial roles in Ca2+-mediated signaling that triggers excitation-contraction coupling, T-lymphocyte activation, fertilization, and many other cellular functions. Three forms of RyR have been identified: RyR1, expressed predominantly in skeletal muscle; RyR2, expressed predominantly in cardiac muscle; and RyR3, expressed in specialized muscles and nonmuscle tissues including the brain. RyR channels are tetramers composed of four subunits each with a molecular mass of approximately 560,000 Da. The tetrameric structures of RyR1 and RyR2 are stabilized by a channel-associated protein known as the FK506 binding protein (FKBP). FKBP is the cytosolic receptor for the immunosuppressant drugs FK506 and rapamycin that inhibit the prolyl isomerase activity of FKBP and can dissociate FKBP from RyRs. Rapamycin and FK506 increase the sensitivity of RyRs to agonists such as caffeine and could be a cause of cardiac dysfunction associated with high-dose immunosuppressant therapy by promoting leakage of Ca2+ from the sarcoplasmic reticulum. The role of prolyl isomerase activity of FKBP in regulating RyR function remains uncertain, and several models have been proposed that could explain how the channel is modulated by its association with FKBP. Three forms of IP3Rs (types 1, 2 and 3) have been characterized by cDNA cloning. Most cells have at least one form of IP3R, and many express all three types. Like RyRs, the IP3R channels are tetramers composed of four subunits (approximately 300,000 Da each). IP3R1 function is regulated by at least two major cellular signaling pathways: the second messenger IP3 activates the channel, and phosphorylation by nonreceptor protein tyrosine kinases (e.g., Fyn) increase its open probability. During end-stage human heart failure, RyR2 mRNA and protein are downregulated, whereas IP3R1 is upregulated, suggesting that altered Ca2+-release channel levels may contribute to defects in Ca2+ homeostasis. Cells that are deficient in IP3R1 exhibit defective T cell-receptor signaling and thus cannot be activated by T cell-receptor stimulation. IP3R1-deficient cells are also resistant to induced apoptosis. Thus RyRs and IP3Rs play critical roles in fundamental and diverse signaling phenomena that include excitation-contraction coupling, T-cell activation, and programmed cell death.

The Type 1 Inositol 1,4,5-Trisphosphate Receptor Gene Is Altered in theopisthotonosMouse

The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.

Pheromone-induced second messenger signaling in the hamster vomeronasal organ.

Compounds present in estrous hamster vaginal discharge modulate male attraction and mounting behavior. These compounds are differentially processed by chemosensory neurons in the main olfactory epithelium (MOE) and vomeronasal organ (VNO). The transduction cascade responsible for this processing is unclear in the VNO, although studies of the MOE suggest that the second messengers cAMP or IP3 may be involved. Here we demonstrate that purified aphrodisin, a hamster mounting pheromone, modulates IP3 production in male VNO membranes without altering cAMP production. Aphrodisin does not alter the concentration of either second messenger in membranes from the MOE. These results confirm the specificity of the VNO in the processing of mounting pheromones and establishes the importance of IP3 cascades in mammalian reproductive behavior.

Subcellular and multicelluar organization of calcium signaling in liver

Cytosolic Ca2+ ([Ca2+]i) is a ubiquitous intracellular messenger in mammalian cells, and imaging studies of single living cells loaded with fluorescent Ca2+ indicators have demonstrated that the [Ca2+]i changes induced by extracellular agonists are often organized in complex temporal and spatial patterns. A number of hormones that regulate hepatic metabolism, including vasopressin and α1adrenergic agonists, bring about their effects through a rise in [Ca2+]i mediated by the Ca2+-mobilizing second messenger IP3. Imaging studies of isolated hepatocytes have demonstrated that the [Ca+]i responses during continuous exposure to these hormones consist of a series of discrete [Ca+]i spikes, the frequency of which is determined by the dose of agonist. By contrast, the amplitude and kinetics of the individual [Ca+]i spikes are not affected by changing the agonist dose. In addition to the temporal organization in the form of [Ca2+]i oscillations, the [Ca2+]i changes are also spatially organized into regenerative [Ca2+]i waves that propagate throughout the cytoplasm and nucleoplasm of the cell from a discrete plasma membrane locus. These oscillatory [Ca2+]i waves are generated by the complex interplay of [Ca2+]i and IP3 in regulating the gating properties of the IP3- receptor Ca2+ channel, which is located in the endoplasmic reticulum Ca2+ store.