Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP₃)-mediated increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_c). Local Ca²⁺ release from an InsP₃ receptor (InsP₃R) cluster present on the sarcoplasmic reticulum is termed a Ca²⁺ puff. Ca²⁺ released via InsP₃R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca²⁺ rise. In smooth muscle, mitochondrial Ca²⁺ uptake maintains global InsP₃-mediated Ca²⁺ release by preventing a negative feedback effect of high [Ca²⁺] on InsP₃R. Mitochondria may regulate InsP₃-mediated Ca²⁺ signals by operating between or within InsP₃R clusters. In the former mitochondria could regulate only global Ca²⁺ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP₃-mediated Ca²⁺ release by operating within (local) or between (global) InsP₃R clusters has been addressed. Ca²⁺ puffs evoked by localized photolysis of InsP₃ in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5–4.0 F/F₀, durations of ∼112 ms at half-maximum amplitude, and were abolished by the InsP₃R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized ΔΨ_M to prevent mitochondrial Ca²⁺ uptake and attenuated Ca²⁺ puffs by ∼66 or ∼60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca²⁺ puffs by ∼62%. The “fast” Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acted like mitochondria to prolong InsP₃-mediated Ca²⁺ release suggesting that mitochondrial influence is via their Ca²⁺ uptake facility. These results indicate Ca²⁺ uptake occurs quickly enough to influence InsP₃R communication at the intra-cluster level and that mitochondria regulate both local and global InsP₃-mediated Ca²⁺ signals.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation

The inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) is an intracellular IP₃-gated calcium (Ca²⁺) release channel and plays important roles in regulation of numerous Ca²⁺-dependent cellular responses. Many intracellular modulators and IP₃R-binding proteins regulate the IP₃R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP₃R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP₃R. The interaction was favored by the cytosolic Ca²⁺ concentration and it functionally inhibited IP₃R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP₃R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP₃R-mediated Ca²⁺ signal through a direct interaction with IP₃R in a cytosolic Ca²⁺-dependent manner.The inositol 1,4,5-trisphosphate (IP₃)³ receptor (IP₃R) consisting of three subtypes, IP₃R1, IP₃R2, and IP₃R3, is a tetrameric intracellular IP₃-gated calcium (Ca²⁺) release channel localized at the endoplasmic reticulum (ER) with its NH₂ terminus and COOH-terminal tail (CTT) exposed to the cytoplasm (1, 2; see Fig. 1A). IP₃Rs are composed of five functional domains. The long NH₂-terminal cytoplasmic region contains three domains, a coupling/suppressor domain, an IP₃-binding core domain, and an internal coupling domain. The COOH-terminal region has a six-membrane spanning channel domain and a short cytoplasmic CTT “gatekeeper domain” that is critical for IP₃R channel opening (2, 3). Ca²⁺ release activity of the IP₃R channel is regulated by many intracellular modulators (ATP, calmodulin, and Ca²⁺), protein kinases, and IP₃R-binding proteins (2, 4), and the tight regulation of IP₃R channel activity by these factors generates various spatial and temporal intracellular Ca²⁺ patterns such as Ca²⁺ spikes and Ca²⁺ oscillations, leading to numerous cellular responses (1, 2, 5, 6).Open in a separate windowFIGURE 1.GIT1 and GIT2 bind to all three subtypes of IP₃R. A, schematic of ER residential IP₃R. The CTT of IP₃R1 is used as bait in a yeast two-hybrid screen. B, schematic representation of GIT1, GIT2, and two GIT1 fragments identified from the yeast two-hybrid screen. Functional domains are indicated. ARF-GAP, ARF-specific GTPase-activating protein domain; ANK-REP, ankyrin repeats; CC, coiled-coil domains; SHD, the Spa2-homology domain; EF, EF-hand; IQ, IQ-like motifs; aa, amino acid. C, GIT1 binds to IP₃R1 in vitro. GST and GST-IP₃R1/CTT were incubated with mouse brain lysate for a pull-down assay. The input and pulled-down samples were probed with α-GIT1. D and E, GIT1 binds to IP₃R1 in vivo. Mouse brain lysates were processed to control IgG and α-IP₃R1 (D) or α-GIT1 (E) for IP. The input and IP samples were probed with α-GIT1 and α-IP₃R1. F and G, both GIT1 and GIT2 bind to all three IP₃R subtypes. HeLa cells coexpressing GFP-fused IP₃R1, IP₃R2, or IP₃R3 and mRFP-fused GIT1 (F) or GIT2 (G) were processed for IP using α-RFP. The input and IP samples were blotted with α-GFP (top) and α-RFP (bottom).One of the physiological roles of IP₃R-mediated Ca²⁺ signaling is a pro-apoptotic regulator during apoptosis. Ca²⁺ released from ER can stimulate several key enzymes activated during apoptosis such as endonucleases (7) and calpain (8). In addition, the close proximity of ER to mitochondria may facilitate the mitochondrial overload of Ca²⁺ released from the IP₃Rs with certain apoptotic stimuli, triggering the opening of the mitochondrial permeability transition pore and the release of apoptotic signaling molecules, such as cytochrome c and apoptosis-inducing factor, which leads to the activation of caspases (5, 6). Moreover, several key components of apoptotic cascades, such as cytochrome c (9) and anti-apoptosis proteins Bcl-2 (10, 11) and Bcl-X_L (12), have been reported to interact with the internal coupling domain and/or the CTT of IP₃R and enhance the Ca²⁺-release activity of IP₃Rs during apoptosis. In this study, we identified the ubiquitously expressed G-protein-coupled receptor kinase-interacting proteins (GIT) (13), GIT1 and GIT2, as novel IP₃R-binding proteins that bind to the CTT of IP₃R and inhibit apoptosis by regulation of IP₃R-mediated Ca²⁺ signal.     

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.     

Redistribution and Increase in Cortical Inositol 1,4,5-Trisphosphate Receptors after Meiotic Maturation of the Mouse Oocyte

Mouse oocytes develop sensitivity to inositol 1,4,5-trisphosphate (IP₃) during oocyte maturation. We recently reported that a change in the organization of the endoplasmic reticulum (ER) during oocyte maturation may contribute to this enhanced sensitivity (Mehlmannet al.,1995,Dev. Biol.170, 607–615). Here, we investigated whether there is an increase in the number of available IP₃receptors after maturation and whether there is a redistribution of IP₃receptors similar to the redistribution of the ER that occurs during maturation. Western blot analysis of the IP₃receptor in oocytes and eggs demonstrated a 1.8-fold increase in immunoreactive mass of the IP₃receptor following oocyte maturation. Microinjection of the function-blocking monoclonal antibody 18A10 inhibited IP₃-induced Ca²⁺release in a concentration-dependent manner in both eggs and oocytes. More antibody was required to inhibit Ca²⁺release to the same extent in eggs compared to oocytes when both were injected with the same concentration of IP₃, suggesting that eggs contain a greater number of functional IP₃receptors. Immunolocalization of the IP₃receptor revealed that receptors were present in large clusters, 1–2 μm in diameter, in the cortex of the mature egg except in a ring-shaped band of cortex adjacent to the meiotic spindle. In contrast, receptor clusters were located around the entire cortex of the immature oocyte and were much smaller (<1 μm); larger patches were sometimes seen, but they did not display the same spherical organization as those in eggs. These results suggest that the number of cortical IP₃receptors increases during mouse oocyte maturation and that this increase may contribute to enhanced Ca²⁺release at fertilization.     

Inositol (1,4,5)-trisphosphate receptor microarchitecture shapes Ca2+ puff kinetics

Inositol (1,4,5)-trisphosphate receptors (IP(3)Rs) release intracellular Ca(2+) as localized Ca(2+) signals (Ca(2+) puffs) that represent the activity of small numbers of clustered IP(3)Rs spaced throughout the endoplasmic reticulum. Although much emphasis has been placed on estimating the number of active Ca(2+) release channels supporting Ca(2+) puffs, less attention has been placed on understanding the role of cluster microarchitecture. This is important as recent data underscores the dynamic nature of IP(3)R transitions between heterogeneous cellular architectures and the differential behavior of IP(3)Rs socialized into clusters. Here, we applied a high-resolution model incorporating stochastically gating IP(3)Rs within a three-dimensional cytoplasmic space to demonstrate: 1), Ca(2+) puffs are supported by a broad range of clustered IP(3)R microarchitectures; 2), cluster ultrastructure shapes Ca(2+) puff characteristics; and 3), loosely corralled IP(3)R clusters (>200 nm interchannel separation) fail to coordinate Ca(2+) puffs, owing to inefficient triggering and impaired coupling due to reduced Ca(2+)-induced Ca(2+) release microwave velocity (<10 nm/s) throughout the channel array. Dynamic microarchitectural considerations may therefore influence Ca(2+) puff occurrence/properties in intact cells, contrasting with a more minimal role for channel number over the same simulated conditions in shaping local Ca(2+) dynamics.     

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-sensitive Ca2+ release across nonvacuolar membranes in cauliflower.

Previous studies have indicated that the vacuole represents the major inositol 1,4,5-trisphosphate (InsP3)-mobilizable Ca2+ pool in higher plants. This findings is in contrast to animal cells, in which the endoplasmic reticulum and plasma membrane constitute the dominant InsP3-sensitive membranes. We used membrane vesicles prepared from cauliflower (Brassica oleracae L.) inflorescences that were separated on continuous sucrose gradients to demonstrate that cauliflower possesses at least two distinct membrane populations that are sensitive to InsP3. One of these membrane populations in nonvacuolar in origin and relies upon a Ca(2+)-ATPase to accumulate Ca2+. In addition, we have shown that two polyclonal antibodies, raised against peptides corresponding to the animal type 1 InsP3 receptor, recognize immunologically related proteins in cauliflower, and that the distribution of immunoreactive proteins on a linear sucrose gradient reinforces the notion that cauliflower contains more than one membrane subtype that is sensitive to InsP3. To our knowledge, this is the first report describing an InsP3-sensitive Ca2+ store other than the vacuole in higher plant cells.     

Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles

The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties.     

Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of oat roots

In plant cells, transient changes in cytoplasmic Ca2+ levels can modulate numerous developmental processes. Ca2+ is accumulated in the vacuole via a H+/Ca2+ antiport system that is energized by the tonoplast H+-pumping ATPase. Inositol 1,4,5-triphosphate (InsP3), but not inositol 1,4-bisphosphate, myo-inositol 1-phosphate, or fructose 2,6-bisphosphate, caused a transient reduction of Ca2+ levels in tonoplast vesicles. The decrease was dependent on InsP3 concentration (Km apparent = 0.6 microM). The InsP3-induced Ca2+ release was blocked by the Ca2+ antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl. These results suggest that the vacuolar membrane is one target site for InsP3 action and that InsP3 may operate as a second messenger in the mobilization of intracellular Ca2+ in plant cells.     

STIM1 Positively Regulates the Ca2+ Release Activity of the Inositol 1,4,5-Trisphosphate Receptor in Bovine Aortic Endothelial Cells

The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca²⁺ signaling that resides in the fact that different signals can be encoded by varying the frequency and the amplitude of the Ca²⁺ response. Cells use both extracellular and intracellular Ca²⁺ pools to modulate the intracellular Ca²⁺ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP₃R), located on the endoplasmic reticulum (ER), is responsible for the release of Ca²⁺ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca²⁺ entry (SOCE). Due to their Ca²⁺ sensor property and their close proximity with IP₃Rs on the ER, STIMs could modulate the activity of IP₃R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP₃R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca²⁺ mobilization in BAECs by a positive effect on both the SOCE and the IP₃R-dependent Ca²⁺ release.     

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.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Inositol (1,4,5)trisphosphate-promoted Ca2+ release from microsomal fractions of rat liver

Crude mitochondrial fractions containing a substantial amount of microsomes accumulate Ca2+ in the presence of ATP, ruthenium red and oligomycin. A proportion of this accumulated Ca2+ is released by the addition of low concentrations (ca. 1 microM) of inositol (1,4,5) trisphosphate . Under some conditions the release is transient, and evidence is presented which suggests that this is due to inhomogeneity in the vesicle population. (1,4,5)inositol trisphosphate -induced Ca2+ release can also be demonstrated, under appropriate experimental conditions, in a more purified microsomal fraction essentially free of mitochondria.     

T-Type Ca2+ Current Activity during Oocyte Growth and Maturation in the Ascidian Styela plicata

Voltage-dependent calcium currents play a fundamental role during oocyte maturation, mostly L-type calcium currents, whereas T-type calcium currents are involved in sperm physiology and cell growth. In this paper, using an electrophysiological and pharmacological approach, we demonstrated, for the first time in oocytes, that T-type calcium currents are present with functional consequences on the plasma membrane of growing immature oocytes of the ascidian Styela plicata. We classified three subtypes of immature oocytes at the germinal vesicle stage on the basis of their size, morphology and accessory cellular structures. These stages were clearly associated with an increased activity of T-type calcium currents and hyperpolarization of the plasma membrane. We also observed that T-type calcium currents oscillate in the post-fertilization embryonic stages, with minimal amplitude of the currents in the zygote and maximal at 8-cell stage. In addition, chemical inhibition of T-type calcium currents, obtained by applying specific antagonists, induced a significant reduction in the rate of cleavage and absence of larval formation. We suggest that calcium entry via T-type calcium channels may act as a potential pacemaker in regulating cytosolic calcium involved in fertilization and early developmental events.     

Inositol 1,3,4,5-tetrakisphosphate increases the duration of the inositol 1,4,5-trisphosphate-mediated Ca2+ transient

The effect of Ins 1,3,4,5-P4 on the intracellular Ca2+ mobilization produced by Ins 1,4,5-P3 has been examined in permeabilized hepatocytes. Ins 1,3,4,5-P4 did not affect the magnitude of the Ins 1,4,5-P3-mediated Ca2+ release but did inhibit re-accumulation of the released Ca2+ back into intracellular stores. This effect was not mimicked by Ins 1,3,4-P3. In hepatocytes, the re-uptake phase of the response results from Ins 1,4,5-P3 hydrolysis. Measurements using labeled substrates indicate that Ins 1,3,4,5-P4 inhibits the hydrolysis of Ins 1,4,5-P3 and vice versa. Since the removal of the 5-phosphate on Ins 1,4,5-P3 and Ins 1,3,4,5-P4 is a common step in the disposal of both compounds, it is suggested that one of the biological effects of Ins 1,3,4,5-P4 may be to slow hydrolysis of Ins 1,4,5-P3 and thereby prolong the duration of a Ca2+ transient.     

Release of Ca2+ from plant hypocotyl microsomes by inositol-1,4,5-trisphosphate

The effect of inositol-1,4,5-trisphosphate on Ca2+ release from microsomes isolated from dark-grown zucchini (Cucurbita pepo L.) hypocotyls was studied. Up to 30% of the Ca2+ taken up by the microsomes in the presence of 2mM ATP, was released by mumolar concentrations of inositol-1,4, 5-trisphosphate. This release was very rapid (less than 0.5 min) and was followed by a slower re-uptake of Ca2+. The microsomal levels of Ca2+ previously attained were not re-established within 5 min. External concentration of free Ca2+ was maintained in the 10(-8)M region during the release. This is the first time that inositol-1,4,5-trisphosphate has been shown to have a regulatory effect on Ca2+ in plant membrane fractions. Phosphoinositides may be important in signal transduction in plant cells, by altering the cytoplasmic Ca2+ activity, a function already known in animal cells.     

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 receptor/Ca2+ channel modulatory role of chromogranin A, a Ca2+ storage protein of secretory granules

The secretory granules of neuroendocrine cells, which contain large amounts of Ca(2+) and chromogranins, have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)), indicating the IP(3)-sensitive intracellular Ca(2+) store role of secretory granules. In our previous study, chromogranin A (CGA) was shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R), at the intravesicular pH 5.5 (Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006). To examine the functional aspect of this coupling, we measured the IP(3)-mediated Ca(2+) release property of the IP(3)R reconstituted into liposomes in the presence and absence of CGA. Presence of CGA in the IP(3)R-reconstituted liposome significantly enhanced the IP(3)-mediated Ca(2+) release from the liposomes. Moreover, the number of IP(3) bound to the reconstituted IP(3)R increased. The fluorescence energy transfer and IP(3)R Trp fluorescence quenching studies indicated that the structure of reconstituted IP(3)R becomes more ordered and exposed in the presence of CGA, suggesting that the coupled CGA in the liposome caused structural changes of the IP(3)R, changing it to a structure that is better suited to IP(3) binding and subsequent Ca(2+) release. These results appear to underscore the physiological significance of IP(3)R-CGA coupling in the secretory granules.