Biomedical subjects

L Missiaen

Publications and source records attributed to L Missiaen.

At least 37 records · Page 2Linked to original sources

PubMed2001

2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trisphosphate receptor, the intracellular Ca2+ pump and the non-specific Ca2+ leak from the non-mitochondrial Ca2+ stores in permeabilized A7r5 cells.

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 the 45Ca2+ uptake into, and on the basal and IP(3)-stimulated unidirectional 45Ca2+ 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 36 microM 2APB, without affecting [3H]IP3 binding 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 91microM 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.

Adenosine Triphosphate↗

PubMed2001

Endothelial intracellular Ca2+ release following monocyte adhesion is required for the transendothelial migration of monocytes.

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 unknown. To characterize intraendothelial signals elicited by leukocyte adhesion and functionally required for their transmigration, we recorded endothelial free cytosolic intracellular Ca(2+)levels ([Ca(2+)]i) during the course of leukocyte adhesion. We show that monocyte and granulocyte adhesion induced Ca(2+)transients in either untreated or TNF-alpha-stimulated microvascular endothelial cells (HMEC-1). The functional significance of these [Ca(2+)]i rises was demonstrated by treating filter-grown endothelial monolayers with BAPTA/AM. This in traendothelial Ca(2+)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 Ca(2+)transients depended on a release from intracellular Ca(2+)stores. Our results thus show that the transmigration of monocytes through endothelial monolayers of microvascular origin is favoured by an increase of the intraendothelial [Ca(2+)]i induced by leukocyte adhesion to the endothelial cells.

Calcium↗

PubMed2001

Calcium puffs are generic InsP(3)-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses.

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.

Adenosine Triphosphate↗

PubMed2000

The Golgi PMR1 P-type ATPase of Caenorhabditis elegans. Identification of the gene and demonstration of calcium and manganese transport.

In recent years, it has been well established that the Ca(2+) 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 Ca(2+) and Mn(2+) 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 gene ATP2C1 cause Hailey-Hailey disease. We report here the identification of the PMR1 homologue in the model organism Caenorhabditis elegans and after ectopic expression the direct study of its ion transport in permeabilized COS-1 cells. The C. elegans genome is predicted to contain a single PMR1 orthologue 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. elegans PMR1 overexpressed in COS-1 cells transports Ca(2+) and Mn(2+) with high affinity into the Golgi apparatus in a thapsigargin-insensitive manner. Part of the accumulated Ca(2+) 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 Ca(2+) store.

Adenosine Triphosphatases↗

PubMed2000

Mapping of the ATP-binding sites on inositol 1,4,5-trisphosphate receptor type 1 and type 3 homotetramers by controlled proteolysis and photoaffinity labeling.

Submillimolar ATP concentrations strongly enhance the inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, by binding specifically to ATP-binding sites on the IP(3) receptor (IP(3)R). To locate those ATP-binding sites on IP(3)R1 and IP(3)R3, both proteins were expressed in Sf9 insect cells and covalently labeled with 8-azido-[alpha-(32)P]ATP. IP(3)R1 and IP(3)R3 were then purified and subjected to a controlled proteolysis, and the labeled proteolytic fragments were identified by site-specific antibodies. Two fragments of IP(3)R1 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 IP(3)R3, only one fragment was labeled. This fragment contained the GXGXXG sequence (amino acids 1920-1925), which is conserved in the three IP(3)R isoforms. The presence of multiple interaction sites for ATP was also evident from the IP(3)-induced Ca(2+) release in permeabilized A7r5 cells, which depended on ATP over a very broad concentration range from micromolar to millimolar.

Adenosine Triphosphate↗

PubMed2000

Effects of the immunosuppressant FK506 on intracellular Ca2+ release and Ca2+ accumulation mechanisms.

The 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. Changes 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. Addition of FK506 to permeabilized SH-SY5Y cells led to a sustained elevation of the medium [Ca2+] corresponding to approximately 30 % of the Ca2+ ionophore A23187-induced [Ca2+] rise. This rise in [Ca2+] was not dependent on mitochondrial activity. This 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 IC50 of 19 microM. The 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. FK506 (10 microM) 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. In 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.

Animals↗

PubMed2000

Epidermal growth factor-mediated targeting of chlorin e6 selectively potentiates its photodynamic activity.

Certain tumor cells, such as squamous carcinoma cells, express an increased number of epidermal growth factor (EGF) receptors. Therefore, we studied the targeted delivery of the photocytotoxic compound Sn-(IV)chlorin e6 monoethylenediamine [SnCe6(ED)] to tumors that overexpress the EGF receptor. EGF was conjugated to SnCe6(ED) through a carrier, such as dextran (Dex) and human serum albumin (HSA), and the photocytotoxicity on the EGF receptor-overexpressing MDA-MB-468 breast adenocarcinoma cell line was evaluated. The photobiological activities of these EGF conjugates, of the conjugates of the photosensitizer to HSA or Dex, or of the photosensitizer alone were compared. The affinity of EGF for its receptor was substantially impaired when conjugated in EGF-Dex-SnCe6(ED), in contrast to EGF-HSA-SnCe6(ED). In corresponding results, EGF-HSA-SnCe6(ED) displayed a high photocytotoxicity (IC50, 63 nM) on MDA-MB-468 cells at a light dose of 27 kJ/m2, whereas EGF-Dex-SnCe6(ED) showed very limited photocytotoxicity. EGF-HSA-SnCe6(ED) was no longer photocytotoxic in the presence of a competing EGF concentration. The high photocytotoxicity of EGF-HSA-SnCe6(ED) was shown to be the result of a high intracellular concentration in MDA-MB-468 cells, which could be lowered dramatically by incubating the conjugate with a competing EGF concentration. In contrast, EGF-Dex-SnCe6(ED) accumulated poorly in MDA-MB-468 cells, in agreement with its low EGF receptor affinity and photocytotoxicity. EGF-HSA-SnCe6(ED) produced much more intracellular reactive oxygen species on light irradiation than EGF-Dex-SnCe6(ED). It is concluded that the photodynamic activity of the EGF-HSA conjugate of SnCe6(ED) on MDA-MB-468 breast adenocarcinoma cells is EGF specific and is much more potent than EGF-Dex-SnCe6(ED) or free SnCe6.

Adenocarcinoma↗

PubMed2000

Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms.

We have expressed the N-terminal 581 amino acids of type 1 myo-inositol 1,4,5-trisphosphate receptor (IP(3)R1), IP(3)R2 and IP(3)R3 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 IP(3)-binding domain and bound IP(3) and adenophostin A with high affinity. Ca(2+) and calmodulin were previously found to maximally inhibit IP(3) binding to lbs-1 by 42+/-6 and 43+/-6% respectively, and with an IC(50) of approx. 200 nM and 3 microM respectively [Sipma, De Smet, Sienaert, Vanlingen, Missiaen, Parys and De Smedt (1999) J. Biol. Chem. 274, 12157-12562]. We now report that Ca(2+) inhibited IP(3) binding to lbs-3 with an IC(50) of approx. 700 nM (37+/-4% inhibition at 5 microM Ca(2+)), while IP(3) binding to lbs-2 was not affected by increasing [Ca(2+)] from 100 nM to 25 microM. Calmodulin (10 microM) inhibited IP(3) binding to lbs-3 by 37+/-4%, while IP(3) binding to lbs-2 was inhibited by only 11+/-2%. The inhibition of IP(3) binding to lbs-3 by calmodulin was dose-dependent (IC(50) approximately 2 microM). We conclude that the IP(3)-binding domains of the various IP(3)R isoforms differ in binding characteristics for IP(3) and adenophostin A, and are differentially modulated by Ca(2+) and calmodulin, suggesting that the various IP(3)R isoforms can have different intracellular functions.

Animals↗

PubMed2000

Differential modulation of inositol 1,4,5-trisphosphate receptor type 1 and type 3 by ATP.

Binding of ATP to the inositol 1,4,5-trisphosphate receptor (IP(3)R) results in a more pronounced Ca(2+)release in the presence of inositol 1,4,5-trisphosphate (IP(3)). Two recently published studies demonstrated a different ATP sensitivity of IP(3)-induced Ca(2+)release in cell types expressing different IP(3)R isoforms. Cell types expressing mainly IP(3)R3 were less sensitive to ATP than cell types expressing mainly IP(3)R1 (Missiaen L, Parys JB, Sienaert I et al. Functional properties of the type 3 InsP(3)receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem 1998;273: 8983-8986; Miyakawa T, Maeda A, Yamazawa T et al. Encoding of Ca(2+)signals by differential expression of IP(3)receptor subtypes. EMBO J 1999;18: 1303-1308). In order to investigate the difference in ATP sensitivity between IP(3)R isoforms at the molecular level, microsomes of Sf9 insect cells expressing full-size IP(3)R1 or IP(3)R3 were covalently labeled with ATP by using the photoaffinity label 8-azido[alpha-(32)P]ATP. ATP labeling of the IP(3)R was measured after immunoprecipitation of IP(3)Rs with isoform-specific antibodies, SDS-PAGE and Phosphorimaging. Unlabeled ATP inhibited covalent linking of 8-azido[alpha-(32)P]ATP to the recombinant IP(3)R1 and IP(3)R3 with an IC(50)of 1.6 microM and 177 microM, respectively. MgATP was as effective as ATP in displacing 8-azido[alpha-(32)P]ATP from the ATP-binding sites on IP(3)R1 and IP(3)R3, and in stimulating IP(3)-induced Ca(2+)release from permeabilized A7r5 and 16HBE14o- cells. The interaction of ATP with the ATP-binding sites on IP(3)R1 and IP(3)R3 was different from its interaction with the IP(3)-binding domains, since ATP inhibited IP(3)binding to the N-terminal 581 amino acids of IP(3)R1 and IP(3)R3 with an IC(50)of 353 microM and 4.0 mM, respectively. The ATP-binding sites of IP(3)R1 bound much better ATP than ADP, AMP and particularly GTP, while IP(3)R3 displayed a much broader nucleotide specificity. These results therefore provide molecular evidence for a differential regulation of IP(3)R1 and IP(3)R3 by ATP.

Adenosine Triphosphate↗

PubMed2000

Abnormal intracellular ca(2+)homeostasis and disease.

A whole range of cell functions are regulated by the free cytosolic Ca(2+)concentration. Activator Ca(2+)from the extracellular space enters the cell through various types of Ca(2+)channels and sometimes the Na(+)/Ca(2+)-exchanger, and is actively extruded from the cell by Ca(2+)pumps and Na(+)/Ca(2+)-exchangers. Activator Ca(2+)can also be released from internal Ca(2+)stores through inositol trisphosphate or ryanodine receptors and is taken up into these organelles by means of Ca(2+)pumps. The resulting Ca(2+)signal is highly organized in space, frequency and amplitude because the localization and the integrated free cytosolic Ca(2+)concentration over time contain specific information. Mutations or functional abnormalities in the various Ca(2+)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 Ca(2+)pumps (Brody disease). Ca(2+)-pump mutations in cutaneous epidermal keratinocytes and cochlear hair cells lead to, skin diseases (Darier and Hailey-Hailey) and hearing/vestibular problems respectively. Mutated Ca(2+)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 Ca(2+)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 Ca(2+)-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 Ca(2+)transporters.

Animals↗

PubMed2000

Calmodulin increases the sensitivity of type 3 inositol-1,4, 5-trisphosphate receptors to Ca(2+) inhibition in human bronchial mucosal cells.

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.

Animals↗

PubMed2000

Receptor-induced Ca(2+) signaling in cultured myenteric neurons.

We studied the effect of excitatory neurotransmitters (10(-5) M) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) of cultured myenteric neurons. ACh evoked a response in 48.6% of the neurons. This response consisted of a fast and a slow component, respectively mediated by nicotinic and muscarinic receptors, as revealed by specific agonists and antagonists. Substance P evoked a [Ca(2+)](i) rise in 68.2% of the neurons, which was highly dependent on Ca(2+) release from intracellular stores, since after thapsigargin (5 microM) pretreatment only 8% responded. The responses to serotonin, present in 90.7%, were completely blocked by ondansetron (10(-5) M), a 5-HT(3) receptor antagonist. Specific agonists of other serotonin receptors were not able to induce a [Ca(2+)](i) rise. Removing extracellular Ca(2+) abolished all serotonin and fast ACh responses, whereas substance P and slow ACh responses were more persistent. We conclude that ACh-induced signaling involves both nicotinic and muscarinic receptors responsible for a fast and a more delayed component, respectively. Substance P-induced signaling requires functional intracellular Ca(2+) stores, and the 5-HT(3) receptor mediates the serotonin-induced Ca(2+) signaling in cultured myenteric neurons.

Acetylcholine↗

PubMed2000

Differential Ca(2+) signaling characteristics of inhibitory and excitatory myenteric motor neurons in culture.

Physiological studies on functionally identified myenteric neurons are scarce because of technical limitations. We combined retrograde labeling, cell culturing, and fluorescent intracellular Ca(2+) concentration ([Ca(2+)](i)) signaling to study excitatory neurotransmitter responsiveness of myenteric motor neurons. 1, 1-Didodecyl-3,3,3',3'-tetramethyl indocarbocyanine (DiI) was used to label circular muscle motor neurons of the guinea pig ileum. DiI-labeled neurons were easily detectable in cultures prepared from these segments. The excitatory neurotransmitters (10(-5) M) acetylcholine, substance P, and serotonin induced a transient rise in [Ca(2+)](i) in subsets of DiI-labeled neurons (66.7, 56.5, and 84. 3%, respectively). DiI-labeled motor neurons were either inhibitory (23.8%) or excitatory (76.2%) as assessed by staining for nitric oxide synthase or choline acetyltransferase. Compared with excitatory motor neurons, significantly fewer inhibitory neurons in culture responded to acetylcholine (0 vs. 69%) and substance P (12.5 vs. 69.2%). We conclude that combining retrograde labeling and Ca(2+) imaging allows identification of differential receptor expression in functionally identified neurons in culture.

Acetylcholine↗

PubMed2000

Mapping of IP(3)-mediated Ca(2+) signals in single human neuroblastoma SH-SY5Y cells: cell volume shaping the Ca(2+) signal.

Fast confocal laser-scanning microscopy was used to study spatiotemporal properties of IP(3)-mediated Ca(2+) release signals in human SH-SY5Y neuroblastoma cells. [Ca(2+)](i) increases were not affected by ryanodine (30 microgM) or caffeine (10 mM) and largely insensitive to removal of external Ca(2+), indicating predominance of IP(3)-induced Ca(2+) release. Ca(2+) signals evoked by high concentration (10 microM) of the muscarinic agonist carbachol appeared as self-propagating waves initiating in cell processes. At low carbachol concentrations (500 nM) Ca(2+) changes in most cells displayed striking spatiotemporal heterogeneity. The Ca(2+) response in the cell body was delayed and had a smaller amplitude and a slower rise time than that in processes. Ca(2+) 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 Ca(2+) release signals.

Caffeine↗

PubMed2000

[Intracellular calcium: physiology and physiopathology].

Many important aspects of our life are regulated by the free cytosolic Ca2+ concentration. The intracellular Ca2+ signal is regulated both in space, frequency and amplitude. Each cell chooses a unique set of Ca2+ signals to control its function. Ca2+ signal transduction is based on rises in free cytosolic Ca2+ concentration. Ca2+ can come from the extracellular space or be released from intracellular stores. Extracellular Ca2+ enters the cell through various types of plasma-membrane Ca2+ channels and leaves the cell using Ca2+ pumps and Na+/Ca(2+)-exchangers. Ca2+ is accumulated in intracellular stores by means of Ca2+ pumps and is released via inositol 1,4,5-trisphosphate (IP3) and ryanodine receptors. Mutations or abnormalities in one of the above mentioned Ca(2+)-transporting proteins can lead to disease. Skeletal-muscle pathology can be caused by abnormal ryanodine receptors (malignant hyperthermia, porcine stress syndrome, central core disease), plasma-membrane Ca2+ channels (hypokalemic periodic paralysis, muscular dysgenesis mice, paraneoplastic Lambert-Eaton myasthenia syndrome) or Ca2+ pumps (Brody disease). Neurologic disorders can be related to altered function of plasma-membrane Ca2+ channels (episodic ataxia type 2, spinocerebellar ataxia type 6, familial hemiplegic migraine, glutamate excitotoxicity, tottering, leaner, lethargic and stargazer mice), IP3 receptors (Lowe's oculocerebrorenal syndrome, manic depression, Alzheimer's disease, opisthotonos mice) and Ca2+ pumps (deafwaddler mouse and wriggle mouse sagami). Two skin diseases are caused by Ca(2+)-pump mutations (Darier disease and Hailey-Hailey disease). Incomplete X-linked congenital stationary night blindness is caused by a mutation in the plasma-membrane Ca2+ channels in rods and cones.

Animals↗

PubMed1999

Cytosolic Ca(2+) controls the loading dependence of IP(3)-induced Ca(2+) release.

It is still debated whether inositol 1,4, 5-trisphosphate(IP(3))-induced Ca(2+) release is loading-dependent. We now report that stimulation of the IP(3) receptor by luminal Ca(2+) depends on the cytosolic [Ca(2+)] in permeabilized A7r5 cells. The EC(50) and maximal extent of Ca(2+) release were loading-dependent in the presence of 5 mM 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid: the EC(50) increased 1.9-fold and the maximal release decreased from 88 to 52% when the stores contained 73% less Ca(2+). In the presence of 0.3 microM free Ca(2+), the EC(50) for filled and less filled stores differed, however, only 1.2-fold and the maximal Ca(2+) release was respectively 96 and 87% of the total releasable Ca(2+). At 1 microM free Ca(2+), the difference in EC(50) between filled and less filled stores again became larger (2.2-fold) and the maximal Ca(2+) release decreased from 93 to 87% when the stores contained less Ca(2+).

Animals↗

PubMed1999

Deletion of the ryanodine receptor type 3 (RyR3) impairs forms of synaptic plasticity and spatial learning.

Deletion of the ryanodine receptor type 3 (RyR3) results in specific changes in hippocampal synaptic plasticity, without affecting hippocampal morphology, basal synaptic transmission or presynaptic function. Robust long-term potentiation (LTP) induced by repeated, strong tetanization in the CA1 region and in the dentate gyrus was unaltered in hippocampal slices in vitro, whereas weak forms of plasticity generated by either a single weak tetanization or depotentiation of a robust LTP were impaired. These distinct physiological deficits were paralleled by a reduced flexibility in re-learning a new target in the water-maze. In contrast, learning performance in the acquisition phase and during probe trial did not differ between the mutants and their wild-type littermates. In the open-field, RyR3(-/-) mice displayed a normal exploration and habituation, but had an increased speed of locomotion and a mild tendency to circular running. The observed physiological and behavioral effects implicate RyR3-mediated Ca(2+) release in the intracellular processes underlying spatial learning and hippocampal synaptic plasticity.

Animals↗

PubMed1999

Nicotinic acid adenine dinucleotide phosphate triggers Ca2+ release from brain microsomes.

Mobilization of Ca2+ from intracellular stores is an important mechanism for generating cytoplasmic Ca2+ signals [1]. Two families of intracellular Ca(2+)-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] [4] [5] [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 IP3 or 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.

Animals↗