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Photodynamic therapy has attracted increasing interest over the last few years, whereby the activation of photosensitizers by light causes the production of reactive oxygen species (ROS), such as singlet oxygen, which are cytotoxic. The goal of our study was to enhance the photodynamic activity of the photosensitizer aluminum phthalocyanine tetrasulfonate (AlPcS4) through its specific delivery to tumor cells. Since many tumor cells, among which are HeLa cells, overexpress the transferrin receptor, we synthesized transferrin conjugated PEG‐liposomes that contained AlPcS4 that could be internalized by receptor mediated endocytosis. The antiproliferative activity of the targeted liposomes was evaluated and compared to the native AlPcS4 and the non‐targeted liposome. These findings were supplemented with data on intracellular concentration of the photo‐active compounds. The accumulation together with ROS production after irradiation was visualized by using confocal microscopy to confirm the data found in the antiproliferative and accumulation assay. Tf‐Lip‐AlPcS4 was 10 times more photocytotoxic (IC50, 0.63 μM) than free AlPcS4 at a light dose of 45 kJ/m whereas Lip‐AlPcS4 displayed no photocytotoxicity at all. The high photocytotoxicity of Tf‐Lip‐AlPcS4 was shown to be the result of a high intracellular concentration (136.5 μM) in HeLa cells, which could be lowered dramatically by incubating the conjugate with a competing transferrin concentration. The images of intracellular accumulation and ROS production matched the accumulation and photocytotoxicity profile of the different photo‐active compounds. The photodynamic activity of the Tf‐Lip‐AlPcS4 conjugate on HeLa cells is much more potent than free AlPcS4 as a result of selective transferrin receptor mediated uptake. © 2002 Wiley‐Liss, Inc.

Human neuroblastoma SH-SY5Y cells, predominantly expressing type 1 inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R), were stably transfected with IP₃R type 3 (IP₃R3) cDNA. Immunocytochemistry experiments showed a homogeneous cytoplasmic distribution of type 3 IP₃Rs in transfected and selected high expression cloned cells. Using confocal Ca²⁺ imaging, carbachol (CCh)-induced Ca²⁺ release signals were studied. Low CCh concentrations (≤750 nM) evoked baseline Ca²⁺ oscillations. Transfected cells displayed a higher CCh responsiveness than control or cloned cells. Ca²⁺ responses varied between fast, large Ca²⁺ spikes and slow, small Ca²⁺ humps, while in the clone only Ca²⁺ humps were observed. Ca²⁺ humps in the transfected cells were associated with a high expression level of IP₃R3. At high CCh concentrations (10 μM) Ca²⁺ transients in transfected and cloned cells were similar to those in control cells. In the clone exogenous IP₃R3 lacked the C-terminal channel domain but IP₃-binding capacity was preserved. Transfected cells mainly expressed intact type 3 IP₃Rs but some protein degradation was also observed. We conclude that in transfected cells expression of functional type 3 IP₃Rs causes an apparent higher affinity for IP₃. In the clone, the presence of degraded receptors leads to an efficient cellular IP₃ buffer and attenuated IP₃-evoked Ca²⁺ release. © 2002 Elsevier Science Ltd

Calmodulin (CaM) is a ubiquitous protein that plays a critical role in regulating cellular functions by altering the activity of a large number of proteins, including the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor (IP3R). CaM inhibits IP3 binding in both the presence and absence of Ca2+ and IP3-induced Ca2+ release in the presence of Ca2+. We have now mapped and characterized a Ca2+-independent CaM-binding site in the N-terminal part of the type 1 IP3R (IP3R1). This site could be responsible for the inhibitory effects of CaM on IP3 binding. We therefore expressed the N-terminal 581 amino acids of IP3R1 as a His-tagged recombinant protein, containing the functional IP3-binding pocket. We showed that CaM, both in the presence and absence of Ca2+, inhibited IP3 binding to this recombinant protein with an IC50 of approx. 2μM. Deletion of the N-terminal 225 amino acids completely abolished the effects of both Ca2+ and CaM on IP3 binding. We mapped the Ca2+-independent CaM-binding site to a recombinant glutathione S-transferase fusion protein containing the first 159 amino acids of IP3R1 and then made different synthetic peptides overlapping this region. We demonstrated that two synthetic peptides matching amino acids 49–81 and 106–128 bound CaM independently of Ca2+ and could reverse the inhibition of IP3 binding caused by CaM. This suggests that these sequences are components of a discontinuous Ca2+-independent CaM-binding domain, which is probably involved in the inhibition of IP3 binding by CaM.

We have investigated how the immunosuppressant drug FK506 affected the basal Ca²⁺ leak in permeabilized A7r5 cells. Non-mitochondrial Ca²⁺ stores loaded to steady state with Ca²⁺ slowly lost their accumulated Ca²⁺ during incubation in a Ca²⁺-free efflux medium. FK506 up to 100 μM had no effect on the basal Ca²⁺ leak. In contrast, the rate of Ca²⁺ release proceeded much faster immediately after washing out FK506. The increase in rate of Ca²⁺ release after washing out of this compound depended on both its initial concentration and on the time of pre-incubation. A similar effect was also observed after removing another immunosuppressant drug (rapamycin) and after removing the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin C. Since all these substances have a high octanol/H₂O partition coefficient and accumulate in the endoplasmic reticulum membrane, we suggest that the transient increase in the basal Ca²⁺ leak is due to the sudden removal of these lipophilic substances from the membrane.

In a previous study we overexpressed the thapsigargin (tg)-insensitive Pmr1 Ca2+pump of the Golgi apparatus of Caenorhabditis elegansin COS-1 cells and studied the properties of the Ca2+store into which it was integrated. Here we assessed the properties of an endogenous tg-insensitive nonmitochondrial Ca2+store in A7r5 and 16HBE14o− cells, which express a mammalian homologue of Pmr1. The tg-insensitive Ca2+store was considerably less leaky for Ca2+than the sarco(endo)plasmic-reticulum Ca2+-ATPase (SERCA)-containing Ca2+store. Moreover like for the worm Pmr1 Ca2+pump expressed in COS-1 cells, Ca2+accumulation into the endogenous tg-insensitive store showed a 2 orders of magnitude lower sensitivity to cyclopiazonic acid than the SERCA-mediated transport. 2,5-Di-(tert-butyl)-1,4-benzohydroquinone was only a very weak inhibitor of the tg-insensitive Ca2+uptake in A7r5 and 16HBE14o− cells and in COS-1 cells overexpressing the worm Pmr1. Inositol 1,4,5-trisphosphate released 11% of the Ca2+accumulated in permeabilized A7r5 cells pretreated with tg with an EC50that was 5 times higher than for the SERCA-containing Ca2+store but failed to release Ca2+in 16HBE14o− cells. In the presence of tg, 15% of intact A7r5 cells responded to 10 μmarginine-vasopressin with a small rise in cytosolic Ca2+concentration after a long latency. In conclusion, A7r5 and 16HBE14o− cells express a Pmr1-containing Ca2+store with properties that differ substantially from the SERCA-containing Ca2+store.

We compared the interaction of the FK506-binding protein (FKBP) with the type 3 ryanodine receptor (RyR3) and with the type 1 and type 3 inositol 1,4,5-trisphosphate receptor (IP3R1 and IP3R3), using a quantitative GST-FKBP12 and GST-FKBP12.6 affinity assay. We first characterized and mapped the interaction of the FKBPs with the RyR3. GST-FKBP12 as well as GST-FKBP12.6 were able to bind ∼30% of the solubilized RyR3. The interaction was completely abolished by FK506, strengthened by the addition of Mg2+, and weakened in the absence of Ca2+but was not affected by the addition of cyclic ADP-ribose. By using proteolytic mapping and site-directed mutagenesis, we pinpointed Val2322, located in the central modulatory domain of the RyR3, as a critical residue for the interaction of RyR3 with FKBPs. Substitution of Val2322for leucine (as in IP3R1) or isoleucine (as in RyR2) decreased the binding efficiency and shifted the selectivity to FKBP12.6; substitution of Val2322for aspartate completely abolished the FKBP interaction. Importantly, the occurrence of the valylprolyl residue as α-helix breaker was an important determinant of FKBP binding. This secondary structure is conserved among the different RyR isoforms but not in the IP3R isoforms. A chimeric RyR3/IP3R1, containing the core of the FKBP12-binding site of IP3R1 in the RyR3 context, retained this secondary structure and was able to interact with FKBPs. In contrast, IP3Rs did not interact with the FKBP isoforms. This indicates that the primary sequence in combination with the local structural environment plays an important role in targeting the FKBPs to the intracellular Ca2+-release channels. Structural differences in the FKBP-binding site of RyRs and IP3Rs may contribute to the occurrence of a stable interaction between RyR isoforms and FKBPs and to the absence of such interaction with IP3Rs.

Elementary Ca(2+) signals, such as "Ca(2+) puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca(2+) signalling. We characterized Ca(2+) puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca(2+) puffs in these cell types suggests that they are a generic elementary Ca(2+) signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca(2+) signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca(2+) puffs. The amplitude and duration of Ca(2+) puffs were substantially reduced. In addition, the number of Ca(2+) puff sites active during the onset of a Ca(2+) wave declined. The consequence of the changes in Ca(2+) puff properties was that cells displayed a lower propensity to trigger regenerative Ca(2+) waves. Therefore, Ca(2+) puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.

Elementary Ca2+ signals, such as ‘Ca2+ puffs’, which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca2+ signalling. We characterized Ca2+ puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca2+ puffs in these cell types suggests that they are a generic elementary Ca2+ signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca2+ signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca2+ puffs. The amplitude and duration of Ca2+ puffs were substantially reduced. In addition, the number of Ca2+ puff sites active during the onset of a Ca2+ wave declined. The consequence of the changes in Ca2+ puff properties was that cells displayed a lower propensity to trigger regenerative Ca2+ waves. Therefore, Ca2+ puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.

Cytosolic Ca2+oscillations can be due to cycles of release and re-uptake of internally stored Ca2+. To investigate the nature of these Ca2+stores, we expressed the Pmr1 Ca2+pump ofCaenorhabditis elegansin COS-1 cells and pretreated the cells with thapsigargin to prevent Ca2+uptake by the sarco(endo)plasmic reticulum Ca2+-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca2+store that was less leaky for Ca2+than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca2+with a regenerative Ca2+signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca2+spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca2+-ATPase. Abortive Ca2+spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca2+store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca2+spiking.

No abstracts.

Although molecular changes accompanying leukocyte extravasation have been investigated intensively, the particular events following leukocyte adhesion and leading to the actual transendothelial migration process remain largely unkown. To characterize in traendothelial signals elicited by leukocyte adhesion and functionally required for their transmigration, we recorded endothelial free cytosolic intracellular Ca2+levels ([Ca2+]i) during the course of leukocyte adhesion. We show that monocyte and granulocyte adhesion induced Ca2+transients in either untreated or TNF-α-stimulated microvascular endothelial cells (HMEC-1). The functional significance of these [Ca2+]irises was demonstrated by treating filter-grown endothelial monolayers with BAPTA/AM. This intraendothelial Ca2+chelation left monocyte adhesion basically unaffected, but caused a significant and dose-dependent reduction of the transendothelial migration of monocytes. Granulocyte diapedesis, on the other hand, was hardly modified. Thapsigargin-treatment of endothelial cells almost completely inhibited the transmigration of monocytes suggesting that the necessary Ca2+transients depended on a release from intracellular Ca2+stores. Our results thus show that the transmigration of monocytes through endothelial monolayers of microvascular origin is favoured by an increase of the intraendothelial [Ca2+]iinduced by leukocyte adhesion to the endothelial cells.

In recent years, it has been well established that the Ca2+concentration in the lumen of intracellular organelles is a key determinant of cell function. Despite the fact that essential functions of the Golgi apparatus depend on the Ca2+and Mn2+concentration in its lumen, little is known on the transport system responsible for ion accumulation. The Golgi ion pump PMR1 has been functionally studied only in yeast. In humans, mutations in the orthologous geneATP2C1cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organismCaenorhabditis elegansand after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. TheC. elegansgenome is predicted to contain a singlePMR1orthologue on chromosome I. We found evidence for alternative splicing in the 5′-untranslated region, but no indication for the generation of different protein isoforms. C. elegansPMR1 overexpressed in COS-1 cells transports Ca2+and Mn2+with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca2+can be released by inositol 1,4,5-trisphosphate, in agreement with the idea that the Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2+store.AJ303081AJ303082

We investigated the interaction of the 12kDa FK506-binding protein (FKBP12) with two ryanodine-receptor isoforms (RyR1 and RyR3) and with two myo-inositol 1,4,5-trisphosphate (IP3) receptor isoforms (IP3R1 and IP3R3). Using glutathione S-transferase (GST)-FKBP12 affinity chromatography, we could efficiently extract RyR1 (42±7% of the solubilized RyR1) from terminal cisternae of skeletal muscle as well as RyR3 (32±4% of the solubilized RyR3) from RyR3-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20µM) but were largely unaffected by RyR-channel modulators. In contrast, neither IP3R1 nor IP3R3 from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and IP3R-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-FKBP12 matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of FKBP12 with RyR1 but not with IP3R1. In order to determine the FKBP12-binding site, we fragmented both RyR1 and IP3R1 by limited proteolysis. We obtained a 45kDa fragment of RyR1 that bound to the GST-FKBP12 matrix, indicating that it retained all requirements for FKBP12 binding. This fragment was identified by its interaction with antibody m34C and must therefore contain its epitope (amino acids 2756–2803). However, no fragment of IP3R1 was retained on the column. These molecular data are in agreement with the lack of correlation between FKBP12 and IP3R1 expression in various cell types. The observation that FKBP12 did not affect IP3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indicated that mature IP3R1 and IP3R3, in contrast to RyR1 and RyR3, did not display a specific, high-affinity interaction with FKBP12.

2-Aminoethoxydiphenyl borate (2APB) is a membrane-permeable blocker of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+release in bi-directional Ca2+-flux conditions. We have now studied the effects of 2APB on the45Ca2+uptake into, and on the basal and IP3-stimulated unidirectional45Ca2+efflux from the non-mitochondrial Ca2+stores in permeabilized A7r5 smooth-muscle cells. 2APB inhibited the IP3-induced Ca2+release, with a half maximal inhibition at 36μM 2APB, without affecting [3H]IP3binding to the receptor. This inhibition did not depend on the IP3, ATP or free Ca2+concentration. The Ca2+pumps of the non-mitochondrial Ca2+stores were half-maximally inhibited at 91μM 2APB. Higher concentrations of 2APB increased the non-specific leak of Ca2+from the stores. We conclude that 2APB can not be considered as a selective blocker of the IP3-induced Ca2+release. Our results can explain the various effects of 2APB observed in intact cells.

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+release, by binding specifically to ATP-binding sites on the IP3receptor (IP3R). To locate those ATP-binding sites on IP3R1 and IP3R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[α-32P]ATP. IP3R1 and IP3R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP3R1 were labeled, each containing one of the previously proposed ATP-binding sites with amino acid sequence GXGXXG (amino acids 1773–1780 and 2016–2021, respectively). In IP3R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920–1925), which is conserved in the three IP3R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP3-induced Ca2+release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.

A whole range of cell functions are regulated by the free cytosolic Ca2+concentration. Activator Ca2+from the extracellular space enters the cell through various types of Ca2+channels and sometimes the Na+/Ca2+-exchanger, and is actively extruded from the cell by Ca2+pumps and Na+/Ca2+-exchangers. Activator Ca2+can also be released from internal Ca2+stores through inositol trisphosphate or ryanodine receptors and is taken up into these organelles by means of Ca2+pumps. The resulting Ca2+signal is highly organized in space, frequency and amplitude because the localization and the integrated free cytosolic Ca2+concentration over time contain specific information. Mutations or functional abnormalities in the various Ca2+transporters, which in vitro seem to induce trivial functional alterations, therefore, often lead to a plethora of diseases. Skeletal-muscle pathology can be caused by mutations in ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central-core disease), dihydropyridine receptors (familial hypokalemic periodic paralysis, malignant hyperthermia, muscular dysgenesis) or Ca2+pumps (Brody disease). Ca2+-pump mutations in cutaneous epidermal keratinocytes and cochlear hair cells lead to, skin diseases (Darier and Hailey-Hailey) and hearing/vestibular problems respectively. Mutated Ca2+channels in the photoreceptor plasma membrane cause vision problems. Hemiplegic migraine, spinocerebellar ataxia type-6, one form of episodic ataxia and some forms of epilepsy can be due to mutations in plasma-membrane Ca2+channels, while antibodies against these channels play a pathogenic role in all patients with the Lambert-Eaton myasthenic syndrome and may be of significance in sporadic amyotrophic lateral sclerosis. Brain inositol trisphosphate receptors have been hypothesized to contribute to the pathology in opisthotonos mice, manic-depressive illness and perhaps Alzheimer’s disease. Various abnormalities in Ca2+-handling proteins have been described in heart during aging, hypertrophy, heart failure and during treatment with immunosuppressive drugs and in diabetes mellitus. In some instances, disease-causing mutations or abnormalities provide us with new insights into the cell biology of the various Ca2+transporters.

1The immunophilin FKBP12 associates with intracellular Ca2+channels and this interaction can be disrupted by the immunosuppressant FK506. We have investigated the effect of FK506 on Ca2+release and Ca2+uptake in permeabilized cell types.2Changes in medium free [Ca2+] were detected by the fluorescent Ca2+indicator fluo‐3 in digitonin‐permeabilized SH‐SY5Y human neuroblastoma cells, DT40 and R23‐11 (i.e. triple inositol 1,4,5‐trisphosphate (IP3) receptor knockout cells) chicken B lymphocytes and differentiated and undifferentiated BC3H1 skeletal muscle cells. 45Ca2+fluxes were studied in saponin‐permeabilized A7r5 rat smooth muscle cells.3Addition of FK506 to permeabilized SH‐SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to ∼30% of the Ca2+ionophore A23187‐induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity.4This FK506‐induced [Ca2+] rise was related to the inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+‐Mg2+‐ATPase (SERCA) Ca2+pump. Oxalate‐facilitated 45Ca2+uptake in SH‐SY5Y microsomes was inhibited by FK506 with an IC50of 19 μm.5The inhibition of the SERCA Ca2+pump was not specific since several macrocyclic lactone compounds (ivermectin > FK506, ascomycin and rapamycin) were able to inhibit Ca2+uptake activity.6FK506 (10 μm) did not affect IP3‐induced Ca2+release in permeabilized SH‐SY5Y and A7r5 cells, but enhanced caffeine‐induced Ca2+release via the ryanodine receptor (RyR) in differentiated BC3H1 cells.7In conclusion, FK506 inhibited active Ca2+uptake by the SERCA Ca2+pump; in addition, FK506 enhanced intracellular Ca2+release through the RyR, but it had no direct effect on IP3‐induced Ca2+release.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP3R) results in a more pronounced Ca2+release in the presence of inositol 1,4,5-trisphosphate (IP3). Two recently published studies demonstrated a different ATP sensitivity of IP3-induced Ca2+release in cell types expressing different IP3R isoforms. Cell types expressing mainly IP3R3 were less sensitive to ATP than cell types expressing mainly IP3R1 (Missiaen L, Parys JB, Sienaert I et al. Functional properties of the type 3 InsP3receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem1998;273: 8983–8986; Miyakawa T, Maeda A, Yamazawa T et al. Encoding of Ca2+signals by differential expression of IP3receptor subtypes. EMBO J1999;18: 1303–1308). In order to investigate the difference in ATP sensitivity between IP3R isoforms at the molecular level, microsomes of Sf9 insect cells expressing full-size IP3R1 or IP3R3 were covalently labeled with ATP by using the photoaffinity label 8-azido[α-32P]ATP. ATP labeling of the IP3R was measured after immunoprecipitation of IP3Rs with isoform-specific antibodies, SDS-PAGE and Phosphorimaging. Unlabeled ATP inhibited covalent linking of 8-azido[α-32P]ATP to the recombinant IP3R1 and IP3R3 with an IC50of 1.6 μM and 177 μM, respectively. MgATP was as effective as ATP in displacing 8-azido[α-32P]ATP from the ATP-binding sites on IP3R1 and IP3R3, and in stimulating IP3-induced Ca2+release from permeabilized A7r5 and 16HBE14o- cells. The interaction of ATP with the ATP-binding sites on IP3R1 and IP3R3 was different from its interaction with the IP3-binding domains, since ATP inhibited IP3binding to the N-terminal 581 amino acids of IP3R1 and IP3R3 with an IC50of 353 μM and 4.0 mM, respectively. The ATP-binding sites of IP3R1 bound much better ATP than ADP, AMP and particularly GTP, while IP3R3 displayed a much broader nucleotide specificity. These results therefore provide molecular evidence for a differential regulation of IP3R1 and IP3R3 by ATP.

Inositol-1,4,5-trisphosphate (IP3) releases Ca2+from intracellular stores by binding to its receptor (IP3R), a multigene family of Ca2+-release channels consisting of IP3R1, IP3R2, and IP3R3. IP3R1 is stimulated by low cytoplasmic Ca2+concentrations and inhibited by high concentrations. Discrepant reports appeared about the effect of cytoplasmic Ca2+on IP3R3, showing either a bell-shaped dependence or only a stimulatory phase with no negative feedback by high Ca2+concentrations. We investigated how calmodulin interfered with the feedback of cytosolic Ca2+on the unidirectional IP3-induced Ca2+release in permeabilized 16HBE14o- bronchial mucosal cells, where IP3R3 represents 93% of the receptors at the mRNA level and 81% at the protein level. Calmodulin inhibited the Ca2+release induced by 1.5 μM IP3with an IC50value of 9 μM. This inhibition was absolutely dependent on the presence of cytosolic Ca2+. Ca2+inhibited the IP3R with an IC50value of 0.92 μM Ca2+in the absence of calmodulin and with an IC50value of 0.15 μM Ca2+in its presence. It is concluded that: 1) IP3R3 can be inhibited by calmodulin, 2) IP3R3 is inhibited by high Ca2+concentrations, and 3) calmodulin shifts the inhibitory part of the Ca2+-response curve toward lower Ca2+concentrations.

Inositol-1,4,5-trisphosphate (IP(3)) releases Ca(2+) from intracellular stores by binding to its receptor (IP(3)R), a multigene family of Ca(2+)-release channels consisting of IP(3)R1, IP(3)R2, and IP(3)R3. IP(3)R1 is stimulated by low cytoplasmic Ca(2+) concentrations and inhibited by high concentrations. Discrepant reports appeared about the effect of cytoplasmic Ca(2+) on IP(3)R3, showing either a bell-shaped dependence or only a stimulatory phase with no negative feedback by high Ca(2+) concentrations. We investigated how calmodulin interfered with the feedback of cytosolic Ca(2+) on the unidirectional IP(3)-induced Ca(2+) release in permeabilized 16HBE14o- bronchial mucosal cells, where IP(3)R3 represents 93% of the receptors at the mRNA level and 81% at the protein level. Calmodulin inhibited the Ca(2+) release induced by 1.5 microM IP(3) with an IC(50) value of 9 microM. This inhibition was absolutely dependent on the presence of cytosolic Ca(2+). Ca(2+) inhibited the IP(3)R with an IC(50) value of 0.92 microM Ca(2+) in the absence of calmodulin and with an IC(50) value of 0.15 microM Ca(2+) in its presence. It is concluded that: 1) IP(3)R3 can be inhibited by calmodulin, 2) IP(3)R3 is inhibited by high Ca(2+) concentrations, and 3) calmodulin shifts the inhibitory part of the Ca(2+)-response curve toward lower Ca(2+) concentrations.

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP3R1), IP3R2 and IP3R3 as recombinant proteins [ligand-binding site 1 (lbs-1), lbs-2, lbs-3] in the soluble fraction of Escherichia coli. These recombinant proteins contain the complete IP3-binding domain and bound IP3 and adenophostin A with high affinity. Ca2+ and calmodulin were previously found to maximally inhibit IP3 binding to lbs-1 by 42±6 and 43±6% respectively, and with an IC50 of approx. 200 nM and 3 μM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca2+ inhibited IP3 binding to lbs-3 with an IC50 of approx. 700 nM (37±4% inhibition at 5 μM Ca2+), while IP3 binding to lbs-2 was not affected by increasing [Ca2+] from 100 nM to 25 μM. Calmodulin (10 μM) inhibited IP3 binding to lbs-3 by 37±4%, while IP3 binding to lbs-2 was inhibited by only 11±2%. The inhibition of IP3 binding to lbs-3 by calmodulin was dose-dependent (IC50≈ 2 μM). We conclude that the IP3-binding domains of the various IP3R isoforms differ in binding characteristics for IP3 and adenophostin A, and are differentially modulated by Ca2+ and calmodulin, suggesting that the various IP3R isoforms can have different intracellular functions.

Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP3-mediated Ca2+release signals in human SH-SY5Y neuroblastoma cells. [Ca2+]iincreases were not affected by ryanodine (30 μM) or caffeine (10 mM) and largely insensitive to removal of external Ca2+, indicating predominance of IP3-induced Ca2+release. Ca2+signals evoked by high concentration (10 μM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca2+changes in most cells displayed striking spatiotemporal heterogeneity. The Ca2+response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca2+changes in processes either occurred in a homogeneous manner throughout the whole process or were sometimes confined to hot spots. Regional differences in surface-to-volume ratio appear to be critical clues that determine the spatiotemporal pattern of intracellular Ca2+release signals.

Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP3-mediated Ca²⁺ release signals in human SH-SY5Y neuroblastoma cells. [Ca²⁺]i increases were not affected by ryanodine (30 μM) or caffeine (10 mM) and largely insensitive to removal of external Ca²⁺, indicating predominance of IP3-induced Ca²⁺ release. Ca²⁺ signals evoked by high concentration (10 μM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca²⁺ changes in most cells displayed striking spatiotemporal heterogeneity. The Ca²⁺ response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca²⁺ changes in processes either occurred in a homogeneous manner throughout the whole process or were sometimes confined to hot spots. Regional differences in surface-to-volume ratio appear to be critical clues that determine the spatiotemporal pattern of intracellular Ca²⁺ release signals.

The amalgamation of 3D extrusion printing (3DEP) and sintering results in a low-cost process compared to other laser-based additive manufacturing techniques. This work used metal injection molding (MIM) raw material of 17–4 PH steel for additive manufacturing. The 3D printing, debinding, and sintering steps were thoroughly evaluated to achieve the highest sintered density. First, the 3DEP of the MIM feedstock was carried out using a screw-based extrusion system at optimum parameters to acquire the high green density and fine surface roughness. The solvent debinding step was carried out on 3D printed samples to remove water-soluble polymer by immersion method. Thermogravimetric analysis was performed to evaluate the decomposition temperature of the backbone material. Further, thermal debinding and sintering steps were conducted in a single step. The thermal debinding temperature was 500 ℃,and the sintering temperatures were chosen as 1100, 1200, 1300 and 1360 ℃. The highest density of ~95.6% was attained at a high sintering temperature. The micro-tomography evaluation was carried out on the 3D printed green and high-density sintered samples to evaluate the internal porosity. The mechanical properties and the microstructure were also evaluated for sintered samples. The work opens a way to fabricate metal complex-shaped parts at low cost using market available MIM feedstock.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.

Abstract: The type-3 inositol 1,4,5-trisphosphate (IP3) receptor, in contrast to the type-1 IP3 receptor (IP3R), is not stimulated by sulfhydryl oxidation and is less sensitive to adenosine 5’-triphosphate. In the present study we compared the effect of pH on the Ca2+ release induced by IP3 and cytosolic Ca2+ between IP3R3-expressing 16HBE14o– cells and IP3R1-expressing A7r5 cells. Changing pH from 6.8 to 7.5 decreased the IP3 concentration required for half-maximal stimulation of IP3R3 (EC50) 10.7-fold (from 2.14 to 0.20 µM). Similar alkalinization decreased the IP3 concentration (EC50) for stimulation of IP3R1 only 2.5-fold (from 0.87 to 0.35 µM). IP3R1 is therefore the more sensitive isoform at pH 6.8, while IP3R3 is more sensitive at pH 7.5. Stimulation and inhibition of IP3R1 and -3 by low and high cytosolic [Ca2+] respectively was observed at both pH 6.8 and 7.5. Increasing [H+] shifted the Ca2+-activation curve of IP3R1 towards higher [Ca2+] but did not affect the Ca2+ dependence of IP3R3. We conclude that IP3R1 and -3 differ markedly in their response to protons.

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) and inositol 1,4,5-trisphosphate receptor (IP3R) isoforms were studied in endothelial cells at the mRNA level by ratio RT–PCR technique and subsequent restriction-enzyme analysis. Three types of cells have been used in the present study: rat adrenal medulla microvascular endothelial cells (RAMEC), rat aortic endothelial cells (RAEC), and human umbilical vein endothelial cells (HUVEC). Our data show the presence of multiple SERCA and IP3R isoforms in each type of endothelial cells. Freshly isolated HUVEC were an exception in this respect since they contained only SERCA3 without SERCA2b messengers. The expression patterns changed upon cell proliferation: SERCA3 and IP3R-1 messengers decreased, while IP3R-3 increased with culturing. Upon cell differentiation, induced by culturing the cells on Matrigel, the expression pattern of the IP3R changed even further in all endothelial cell types: IP3R-1 was reduced in all three cell kinds, while IP3R-3 raised significantly in RAEC and RAMEC. In HUVEC the expression of SERCA returned, upon differentiation, to the levels observed in the freshly isolated cells. Thus, the plasticity of expression of various SERCA and IP3R isoforms shows that possibly different Ca2+pools may play distinct roles in cell proliferation and differentiation.

Xestospongins, a group of macrocyclic bis-1-oxaquinolizidines isolated from the Australian sponge,Xestospongiaspecies, are potent blockers of the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+release in bi-directional Ca2+-flux conditions. We have now studied the effects of xestospongin C on the45Ca2+uptake and the uni-directional45Ca2+efflux in permeabilized A7r5 smooth-muscle cells. Xestospongin C not only inhibits the IP3-induced Ca2+release, but is also an equally potent blocker of the endoplasmic-reticulum Ca2+pump, while it has no effect on the passive Ca2+leak. The inhibition of the IP3receptor did not depend on the IP3, Ca2+or ATP concentration. Xestospongin C can, therefore, not be considered as a selective blocker of IP3receptors.

Mobilization of Ca2+from intracellular stores is an important mechanism for generating cytoplasmic Ca2+signals [1]. Two families of intracellular Ca2+-release channels – the inositol-1,4,5-trisphosphate (IP3) receptors and the ryanodine receptors (RyRs) – have been described in mammalian tissues [2]. Recently, nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule derived from NADP+, has been shown to trigger Ca2+release from intracellular stores in invertebrate eggs [3–6]and pancreatic acinar cells [7]. The nature of NAADP-induced Ca2+release is unknown but it is clearly distinct from the IP3- and cyclic ADP ribose (cADPR)-sensitive mechanisms in eggs (reviewed in [8,9]). Furthermore, mammalian cells can synthesize and degrade NAADP, suggesting that NAADP-induced Ca2+release may be widespread and thus contribute to the complexity of Ca2+signalling [10,11]. Here, we show for the first time that NAADP evokes Ca2+release from rat brain microsomes by a mechanism that is distinct from those sensitive to IP3or cADPR, and has a remarkably similar pharmacology to the action of NAADP in sea urchin eggs [12]. Membranes prepared from the same rat brain tissues are able to support the synthesis and degradation of NAADP. We therefore suggest that NAADP-mediated Ca2+signalling could play an important role in neuronal Ca2+signalling.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+release by calmodulin at various Ca2+concentrations, we have studied how cytosolic Ca2+and Sr2+interfere with the effects of calmodulin on the IP3-induced Ca2+release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+release through the IP3receptor with an IC50of 4.6 μmif the cytosolic Ca2+concentration was 0.3 μmor higher. This inhibition was particularly pronounced at low IP3concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+release if the cytosolic Ca2+concentration was below 0.3 μm. Calmodulin also inhibited Ca2+release through the IP3receptor in the presence of at least 10 μmSr2+. We conclude that cytosolic Ca2+or Sr2+are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.

It is still debated whether inositol 1,4,5-trisphosphate(IP3)-induced Ca2+release is loading-dependent. We now report that stimulation of the IP3receptor by luminal Ca2+depends on the cytosolic [Ca2+] in permeabilized A7r5 cells. The EC50and maximal extent of Ca2+release were loading-dependent in the presence of 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid: the EC50increased 1.9-fold and the maximal release decreased from 88 to 52% when the stores contained 73% less Ca2+. In the presence of 0.3 μM free Ca2+, the EC50for filled and less filled stores differed, however, only 1.2-fold and the maximal Ca2+release was respectively 96 and 87% of the total releasable Ca2+. At 1 μM free Ca2+, the difference in EC50between filled and less filled stores again became larger (2.2-fold)and the maximal Ca2+release decreased from 93 to 87% when the stores contained less Ca2+.

Abstract:  There is still no agreement on the mechanism of the intracellular action of low concentrations of inositol 1,4,5-trisphosphate (IP3). Intracellular Ca2+ stores may transiently release some Ca2+ before they become insensitive to IP3. Alternatively, stores with a low IP3 threshold may lose all their Ca2+ and the others none. We now report that the IP3 threshold was not correlated with the extent of Ca2+ release in permeabilized A7r5 smooth-muscle cells. In contrast, the maximum rate of release, which was changed either by varying the level of IP3 receptor (IP3R) activation, or by changing the concentration of IP3R at a constant level of IP3R activation, was directly related to the extent of Ca2+ release. We conclude that IP3-induced Ca2+ release reflects partial emptying of the stores and not all-or-none Ca2+ release of separate quanta.

Ca2+ release from intracellular stores occurs via two families of intracellular channels, each with their own specific agonist: Ins(1,4,5)P3 for the Ins(1,4,5)P3 receptor and cyclic ADP-ribose (cADPR) for the ryanodine receptor. We now report that cADPR inhibited Ins(1,4,5)P3-induced Ca2+ release in permeabilized A7r5 cells with an IC50 of 20 μM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 μM. This inhibition was accompanied by an increase in specific [3H]Ins(1,4,5)P3 binding. 8-Amino-cADPR, but not 8-bromo-cADPR, antagonized this effect of cADPR. The inhibition was prevented by a whole series of inositol phosphates (10 μM) that did not affect Ins(1,4,5)P3-induced Ca2+ release, and by micromolar concentrations of PPi and various nucleotide di- or triphosphates. We propose that cADPR must interact with a novel regulatory site on the Ins(1,4,5)P3 receptor or on an associated protein. This site is neither the Ins(1,4,5)P3-binding domain, which prefers Ins(1,4,5)P3 and only binds nucleotides and PPi in the millimolar range, nor the stimulatory adenine nucleotide binding site.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.

The apparent Mg2+-activated ATPase activity measured by the continuous NADH-coupled enzyme assay was studied in a number of microsomal preparations obtained from smooth muscle of the myometrium from pregnant or 17 beta-oestradiol-pretreated rats, the bovine aorta, the guinea-pig taenia coli, the rabbit ear artery and pig antrum. It was shown that this ATPase assay is prone to the effects of a number of artefacts that are tissue-dependent. The apparent Mg2+-ATPase activity in microsomes (microsomal fractions) from myometrium, aorta and taenia coli declines non-linearly during the assay. Its initial high rate gradually diminishes over 15-60 min, depending on the type of smooth muscle, to a constant value. This decline depends on the presence of ATP and can be partially prevented by concanavalin A. The non-linearity is limited in microsomes from rabbit ear artery. In microsomes from antrum the apparent Mg2+-ATPase activity actually increases with time, albeit gradually. Storage on ice of the microsomes of the aorta, and especially of myometrium of pregnant rats and of taenia coli, is accompanied over a few hours after their preparation by a gradual suppression of the component of the Mg2+-ATPase activity that is inhibited by ATP. The Mg2+-ATPase activity in microsomes from antrum remains constant. NADH oxidase activity accounts for 10% of the Mg2+-ATPase activity in microsomes from stomach smooth muscle. The apparent initial non-linearity of the Mg2+-ATPase activity in that tissue is due to a time-dependent decrease of a rotenone-sensitive NADH oxidase activity. The adenylate kinase activity, as deduced from the effect of the adenylate kinase inhibitor P1,P5-di(adenosine-5′) pentaphosphate, could account for 45.0, 35.0 and 31.0% respectively of the Mg2+-ATPase activity in microsomes from stomach, myometrium and aorta. No adenylate kinase activity could be detected in microsomes from ear artery and taenia coli. When microsomes from stomach smooth muscle were separated on a sucrose gradient, the contribution of adenylate kinase and NADH oxidase to the Mg2+-ATPase activity was most pronounced in the higher-density fractions. Part of the NADH oxidase activity and of the Mg2+-ATPase activity, and most of the adenylate kinase activity, are not sedimented at 224000 gmax. for 30 min and may therefore be present as soluble enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

We developed a unidirectional 45Ca2+efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP3), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP3action, which was around 32 nM InsP3under control conditions. The InsP3-induced Ca2+release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP3concentrations. Stimulatory concentrations of thimerosal (10 μM) shifted the threshold to 4.5 nM InsP3and increased both the cooperativity and the maximal normalized rate of Ca2+release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 μM) but then the effects on the cooperativity and on the normalized rate of Ca2+release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP3concentrations. Luminal Ca2+did not affect the threshold for InsP3action but stimulated the normalized release at each InsP3concentration. The inhibitory effect of 10 μM free cytosolic Ca2+was associated with a shift in threshold to higher InsP3concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP3action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP3receptor.

1. The properties of intracellular Ca2+ stores of intact‐ and of saponin‐skinned A7r5 (an established cell line from embryonic rat aorta) smooth muscle cells were studied by measuring 45Ca2+ and 54Mn2+ fluxes. 2. Application of 5 microM‐vasopressin to intact cells increased the fractional loss of 45Ca2+ in Ca2(+)‐free solution by a factor of 5.2. This effect was not influenced by a pre‐incubation with 10 microM‐ryanodine. Caffeine (25 mM) did not stimulate the fractional loss of 45Ca2+ from intact cells. 3. In skinned cells 10 microM‐IP3 (inositol 1,4,5‐trisphosphate) and 5 microM‐A23187 (a calcium ionophore) released the same amount of 45Ca2+. This release did not require GTP and was not affected by a pre‐incubation with 10 microM‐ryanodine. Caffeine (25 mM) did not release stored Ca2+. 4. NaF (1 mM) plus 10 microM‐AlCl3 inhibited by 72% the 45Ca2+ uptake by the IP3‐sensitive store of skinned cells at 0.15 microM‐Ca2+. Cyclic AMP‐dependent protein kinase did not stimulate this ATP‐dependent 45Ca2+ uptake, nor could the presence of phospholamban be demonstrated immunologically. 5. The 45Ca2+ uptake by cells which had been depleted of Ca2+ with 5 microM‐vasopressin was 69% higher than the uptake obtained without such proceeding depletion. This enhanced 45Ca2+ uptake did not occur through voltage‐operated Ca2+ channels, because blockade of these channels with verapamil, or depolarization of the plasma membrane by increasing [K+] from 5.9 to 59 mM in the presence of verapamil, did not modify this uptake. 6. A similar increase of the 54Mn2+ uptake occurred in intact cells with a depleted Ca2+ store. If, however, the cells were first skinned and subsequently exposed to 54Mn2+, the ATP‐dependent 54Mn2+ uptake amounted to less than 6% of the ATP‐dependent 45Ca2+ uptake. 7. If intact cells were first exposed to a 45Ca2(+)‐ or 54Mn2(+)‐containing solution, and subsequently skinned in a non‐radioactive intracellular solution, the addition of 10 microM‐A23187 to these cells released stored Ca2+ or Mn2+. The amount of released Ca2+ was only slightly larger than the amount of released Mn2+. If the intracellular store was depleted before loading, the amount of Ca2+ or Mn2+ released by the ionophore increased by 68 and 28%, respectively. 8. It is concluded that A7r5 smooth muscle cells do not express a Ca2(+)‐induced Ca2+ release mechanism, but do contain an IP3‐induced Ca2+ release mechanism which can release approximately all intracellularly accumulated 45Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Spontaneous Ca2+ release from the inositol 1,4,5‐trisphosphate (InsP3)‐sensitive stores in permeabilized hepatocytes was monitored using Fluo‐3 to measure the free [Ca2+] of the medium bathing the cells. 2. Permeabilized cells rapidly sequestered Ca2+, reducing the [Ca2+] to 103 +/‐ 5 nM. Under conditions that depended critically upon cell density and the amount of Ca2+ in the medium, this was followed by a slow increase in [Ca2+] culminating in a substantial Ca2+ spike representing synchronous discharge from the InsP3‐sensitive stores. 3. During the latency preceding the Ca2+ spike, the stores increased their sensitivity to InsP3. This sensitization seemed to be an all‐or‐none phenomenon. 4. Oxidized glutathione and thimerosal promoted the spontaneous release by sensitizing the InsP3 receptor. 5. An increase in the [Ca2+] within the stores was required for both the increased sensitivity to InsP3 and the subsequent spike. 6. Caffeine (6 mM) antagonized the effect of very low InsP3 concentrations and abolished the Ca2+ spike, without itself releasing Ca2+. 7. Our results suggesting that luminal Ca2+ may sensitive InsP3‐sensitive stores leading to spontaneous Ca2+ mobilization will be discussed in the light of a modified version of the two‐pool model for explaining cytosolic Ca2+ oscillations.

There is no consensus about the different types of Ca2+ transport processes in the endoplasmic reticulum that are targeted by the sulphydryl reagent thimerosal. We have therefore investigated how thimerosal affects the various Ca2+ transport processes in permeabilized A7r5 smooth-muscle cells, using an unidirectional 45Ca2+ flux technique. Thimerosal up to a concentration of 32 µM did not have an effect on the passive 45Ca2+ leak from the stores, while higher concentrations increased this aspecific leak. Thimerosal inhibited the endoplasmic reticulum Ca2+ pump with an EC50 of 9 µM. Thimerosal exerted a biphasic effect on the Ca2+ release induced by inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] with a stimulation of the release at thimerosal concentrations below 10 µM, and an inhibitory effect at higher concentrations. Thimerosal (2.5–250 µM) did not exert an effect on the specific binding of [3H]Ins(1,4,5)P3 to its receptor, indicating that it probably did not act at the level of the binding site. This finding contrasts with the effect of the closely related sulphydryl reagent parachloromercuriphenylsulphonate, which, at high concentrations, inhibited [3H]Ins(1,4,5)P3 binding. The effects of thimerosal were largely prevented by the sulphydryl reducing agent dithiothreitol (3 mM). We conclude that thimerosal concentrations ranging from 0.32 to 1 µM can stimulate the Ins(1,4,5)P3-induced Ca2+ release without inhibiting the Ca2+ pumps or without increasing the passive Ca2+ permeability of the endoplasmic reticulum.

We have investigated whether reducing agents and substances that interfere with glutathione metabolism would affect the histamine-induced rises in internal Ca2+ concentration ([Ca2+]i) in indo-1-loaded HeLa cells. Individual cells responded to 1 µM histamine with either baseline or sinusoidal Ca2+ oscillations, a single Ca2+ peak or a maintained elevation of the [Ca2+]i. Only a few cells did not respond. The sulphydryl reducing agent dithiothreitol (5 mM) did not affect these responses to histamine. A 24-h preincubation with 1 mM dl-buthionine (SR)-sulphoximine, which reduces the cellular glutathione content to less than 20% of its control value, affected neither these histamine responses, nor the [Ca2+]i rises after application of 2 µM thapsigargin. We conclude that oxidation of critical sulphydryl groups is not required for the normal response to histamine and also that glutathione plays no role in agonistinduced Ca2+ signalling in HeLa cells.

Intracellular Ca2+ signals in response to inositol 1,4,5-trisphosphate-producing agents often present themselves as Ca2+ oscillations and propagating Ca2+ waves originating at discrete initiation sites. We studied the spatial organization of the Ca2+ signal in single CPAE endothelial cells stimulated with adenosine triphosphate. The long, thin processes presented a higher agonist sensitivity and, for the same agonist concentration, a faster rise in cytoplasmic Ca2+ concentration and rate of wave propagation than the cell body. Ca2+ waves originated preferentially in one of these processes and then invaded the cell body. Removal of external Ca2+ induced a progressive inhibition up to blockade of the response in the process but not in the cell body. These findings suggest that CPAE cells contain many individual store units, each of which has the inherent ability to set the stage for Ca2+ release. A diffusing messenger originating from the initiation zone then coordinates the events leading to Ca2+ release in the individual store units to produce a Ca2+ wave.

Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012). Strong 45Ca2+ and ruthenium red binding domains were localized in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains were located within the InsP3-binding domain, and three Ca2+ binding stretches were localized in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca2+ and Sr2+ on the EC50 and cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+ was due to a shift in EC50 toward lower InsP3 concentrations, and this effect was mimicked by Sr2+. The inhibition by cytosolic Ca2+ was caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on the cooperativity occurred at lower Ca2+ concentrations than the inhibitory effect on the EC50. In addition, Sr2+ mimicked the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca2+ stimulated the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. We conclude that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+.

The binding of inositol 1,4,5-trisphosphate (IP3) to the IP3receptor (IP3R) is modulated by various compounds. Until now, limited progress has been made concerning the isoform-specific effects of these modulators. In this study, we examined how [3H]IP3binding to the three IP3R isoforms is modulated by cyclic ADP-ribose (cADPR) and by the SH-reagent thimerosal. We used rabbit cerebellum, RBL-2H3 rat mucosal mast cells and 16HBE14o- human bronchial epithelial cells as model systems for IP3R-1, -2 and -3 respectively. [3H]IP3binding was first characterized at various pH values. We showed that [3H]IP3binding to RBL–2H3 microsomes was more enhanced by increasing the pH from 7.4 to 8.3 than that to rabbit cerebellar microsomes. In contrast, [3H]IP3binding to 16HBE14o- microsomes was not stimulated at alkaline pH. At pH 7.4, cADPR (50 μM) increased [3H]IP3binding to rabbit cerebellar microsomes, RBL–2H3 and 16HBE14o- microsomes 1.5-fold, 1.3-fold and 1.8-fold respectively. The effect of cADPR on IP3binding was abolished at pH 8.3. Scatchard analysis indicated that cADPR induced in cerebellum a decrease in IP3affinity (KDincreases from 150 nM to 252 nM) of the IP3R and a parallel increase in Bmax(from 4.8 pmol/mg to 11.1 pmol/mg). Thimerosal dose-dependently increased [3H]IP3binding to rabbit cerebellar microsomes. The stimulatory effects of cADPR and thimerosal were not additive. Binding to cerebellar microsomes returned to control level in the presence of 500 μM thimerosal. In contrast, thimerosal (up to 500 μM) had no stimulatory effect and only a very slight, if any, inhibitory effect on [3H]IP3binding to RBL–2H3 and 16HBE14o- microsomes respectively. These results indicate that IP3binding to the IP3R isoforms can be differentially modulated by cADPR and thimerosal.

The type-3 inositol 1,4,5-trisphosphate (InsP3) receptor is the major isoform expressed in 16HBE14o− cells from bronchial mucosa, representing 93% at the mRNA level as determined by ratio reverse transcription-polymerase chain reaction and about 81% at the protein level as determined with isoform-specific antibodies (Sienaert, I., Huyghe, S., Parys, J. B., Malfait, M., Kunzelmann, K., De Smedt, H., Verleden, G. M., and Missiaen, L., Pflügers Arch. Eur. Y. Physiol., in press). The present 45Ca2+efflux experiments indicate that these InsP3receptors were 3 times less sensitive to InsP3and 11 times less sensitive to ATP than those in A7r5 cells, where the type-1 InsP3receptor is the main isoform. ATP did not increase the cooperativity of the InsP3-induced Ca2+release in 16HBE14o− cells, in contrast to its effect in A7r5 cells. The sulfhydryl reagent thimerosal also did not stimulate InsP3-induced Ca2+release in 16HBE14o− cells, again in contrast to its effect in A7r5 cells. Adenophostin A was more potent than InsP3in stimulating the release in both cell types. The biphasic activation of the InsP3receptor by cytosolic Ca2+occurred in both cell types. We conclude that Ca2+release mediated by the type-3 InsP3receptor mainly differs from that mediated by the type-1 InsP3receptor by its lack of stimulation by sulfhydryl oxidation and its lower ATP and InsP3sensitivity. The predominant expression of the type-3 InsP3receptor in the bronchial mucosa may be part of a mechanism coping with oxidative stress in that tissue.

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores is generally assumed to be a ‘quantal’ process because low InsP3 concentrations mobilize less Ca2+ than high concentrations and a submaximal concentration does not release all the InsP3-mobilizable Ca2+. However, some recent reports questioned the generally accepted view that a low dose of InsP3 is unable to empty the whole store. We have now challenged the stores of permeabilized A7r5 cells in InsP3 for much longer periods than previously reported, to assess directly whether the slow phase of the release would empty the whole store (a non-quantal response) or only a fraction of it (a quantal response). Addition of a maximal [InsP3] at the end of a prolonged (92 min) stimulation with a submaximal [InsP3] resulted in additional Ca2+ release. Experiments in which the stores were challenged with different submaximal InsP3 concentrations for long time periods revealed that a lower [InsP3] never released the same amount of Ca2+ as a higher [InsP3]. This quantal pattern of Ca2+ release occurred both at 25 °C and at 4 °C. There was a time-dependent increase in the fraction of Ca2+ recruited by the lower compared with the higher [InsP3]. This recruitment of Ca2+ persisted if the [InsP3] was decreased, but was largely prevented by palmitoyl-CoA, a potent blocker of the luminal Ca2+ translocation between individual store units. ATP, in the presence of InsP3, released Ca2+ under conditions permitting the recruitment of no additional InsP3 receptors, indicating that an all-or-none emptying of a fraction of the stores cannot be the only mechanism responsible for quantal Ca2+ release in A7r5 cells. We conclude that some of the previously published evidence for a non-quantal Ca2+ release pattern should be reinterpreted.

Previous reports suggested the expression of four or five different Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] isoforms in mouse cells [Ross, Danoff, Schell, Snyder and Ullrich (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4265–4269; De Smedt, Missiaen, Parys, Bootman, Mertens, Van Den Bosch and Casteels (1994) J. Biol. Chem. 269, 21691–21698]. To explore this diversity further, we have isolated and sequenced partial clones of two Ins(1,4,5)P3R mRNAs from the mouse embryonic C3H10T½ cell line. These clones showed between 94.2 and 94.9% sequence identity with the corresponding rat Ins(1,4,5)P3R-II and Ins(1,4,5)P3R-III isoforms. Based on these newly obtained sequences we have determined the relative expression of the different Ins(1,4,5)P3R mRNAs in cultured cells and in animal tissues of mouse origin by a ratio reverse transcriptase polymerase chain reaction (RT-PCR). Ins(1,4,5)P3R-I was very prominent in brain and cerebellum and Ins(1,4,5)P3R-II in epithelia such as kidney as well as in both cardiac and skeletal muscle. Ins(1,4,5)P3R-III was highly expressed in all cultured cell types and in tissues with high cell turnover, e.g. testis. The prominent expression of Ins(1,4,5)P3R-I and Ins(1,4,5)P3R-III in A7r5 and C3H10T½ cells respectively was confirmed by immunoblot analysis and was compatible with a lower threshold for Ins(1,4,5)P3-induced Ca2+ release in the former cell type. Screening of a large number of mouse cell lines and tissues revealed the presence of Ins(1,4,5)P3R-I as well as of the Ins(1,4,5)P3R-II and Ins(1,4,5)P3R-III isoforms which were identified in the present study, but in contrast with previous reports there was no evidence for more isoform diversity.