Biomedical subjects

L Missiaen

Publications and source records attributed to L Missiaen.

At least 55 records · Page 3Linked to original sources

PubMed1999

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

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.

Animals↗

PubMed1999

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin.

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.

Animals↗

PubMed1999

IP3-induced Ca2+ release in A7r5 vascular smooth-muscle cells represents a partial emptying of the stores and not an all-or-none Ca2+ release of separate quanta.

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.

Adenosine Triphosphate↗

PubMed1999

The relative order of IP3 sensitivity of types 1 and 3 IP3 receptors is pH dependent.

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 microM). Similar alkalinization decreased the IP3 concentration (EC50) for stimulation of IP3R1 only 2.5-fold (from 0.87 to 0.35 microM). 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.

Animals↗

PubMed1999

Calcium handling proteins in isolated spinal motoneurons.

Amyotrophic lateral sclerosis is characterized by motoneuron degeneration, in which glutamate-induced cell death is thought to play a pathogenic role. This excitotoxic process is mediated by cytosolic Ca2+ overload. The glutamatergic ionotropic channel molecules, which constitute a major route of Ca2+ entry, were present on cultured spinal motoneurons. Using ratio RT-PCR, the relative presence in isolated motoneurons of the GluR subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor was evaluated. GluR1 and GluR2 mRNAs were present abundantly, while GluR3 and GluR4 mRNAs were much less abundant. The relative amount of mRNAs encoding the different protein isoforms responsible for Ca2+ uptake into the internal stores and for controlled release of Ca2+ from these stores was also determined. For the sarco/endoplasmic reticulum Ca2+ ATPases (SERCAs), only the SERCA2b class 4 splice variant was found. The inositol 1,4,5-trisphosphate receptor (IP3R) mRNAs were mainly transcribed from the IP3RI and IP3RII genes. Heterogeneity was also observed for the ryanodine receptors (RyR) as the RyR1, RyR2 and RyR3 mRNAs were present.

Animals↗

PubMed1999

Modulation of type 1, 2 and 3 inositol 1,4,5-trisphosphate receptors by cyclic ADP-ribose and thimerosal.

The binding of inositol 1,4,5-trisphosphate (IP3) to the IP3 receptor (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]IP3 binding 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]IP3 binding was first characterized at various pH values. We showed that [3H]IP3 binding 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]IP3 binding to 16HBE14o- microsomes was not stimulated at alkaline pH. At pH 7.4, cADPR (50 microM) increased [3H]IP3 binding 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 IP3 binding was abolished at pH 8.3. Scatchard analysis indicated that cADPR induced in cerebellum a decrease in IP3 affinity (KD increases 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]IP3 binding 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 microM thimerosal. In contrast, thimerosal (up to 500 microM) had no stimulatory effect and only a very slight, if any, inhibitory effect on [3H]IP3 binding to RBL-2H3 and 16HBE14o- microsomes respectively. These results indicate that IP3 binding to the IP3R isoforms can be differentially modulated by cADPR and thimerosal.

Adenosine Diphosphate Ribose↗

PubMed1999

Adenine-nucleotide binding sites on the inositol 1,4,5-trisphosphate receptor bind caffeine, but not adenophostin A or cyclic ADP-ribose.

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). We have expressed the cDNAs encoding two putative adenine-nucleotide binding sites of the neuronal form of IP3R-1 as glutathione S-transferase (GST)-fusion proteins in bacteria. Specific [alpha-32P]ATP binding was observed for the two GST-fusion proteins, representing aa 1710-1850 and aa 1944-2040 of IP3R-1. The ATP-binding sites in both fusion proteins had the same nucleotide specificity as found for the intact IP3R (ATP > ADP > AMP > GTP). Smaller GST-fusion proteins (aa 1745-1792 and aa 2005-2023) displayed a much weaker ATP-binding activity. CoA, which also potentiated IP3-induced Ca2+ release in A7r5 cells, interacted with the ATP-binding sites on the fusion proteins. Such interaction was not observed for 1,N6-etheno CoA and 3'-dephospho-CoA, which are much less effective in potentiating IP3-induced Ca2+ release. Since the adenine-containing compounds adenophostin A, caffeine and cyclic ADP-ribose modulate IP3-induced Ca2+ release, a possible effect of these compounds on the ATP-binding sites was examined. ATP stimulated adenophostin A- and IP3-induced Ca2+ release in A7r5 cells with an EC50 of respectively 21 and 20 microM. Also the threshold concentration of ATP for stimulating the release was similar for the two agonists. Adenophostin A (100 microM) and cyclic ADP-ribose (100 microM) were ineffective in displacing [alpha-32P]ATP from the binding sites of both GST-fusion proteins. Caffeine (50 mM), however, inhibited [alpha-32P]ATP binding to both fusion proteins by more than 50%. These data provide evidence for a direct interaction of caffeine but not of adenophostin A or cyclic ADP-ribose with the adenine-nucleotide binding sites of the IP3R.

Adenine↗

PubMed1999

Expression patterns of sarco/endoplasmic reticulum Ca(2+)-ATPase and inositol 1,4,5-trisphosphate receptor isoforms in vascular endothelial cells.

Expression patterns of sarcoplasmic/endoplasmic-reticulum Ca(2+)-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.

Adrenal Medulla↗

PubMed1998

Functional properties of the type-3 InsP3 receptor in 16HBE14o- bronchial mucosal cells.

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 InsP3 receptors were 3 times less sensitive to InsP3 and 11 times less sensitive to ATP than those in A7r5 cells, where the type-1 InsP3 receptor 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 InsP3 in stimulating the release in both cell types. The biphasic activation of the InsP3 receptor by cytosolic Ca2+ occurred in both cell types. We conclude that Ca2+ release mediated by the type-3 InsP3 receptor mainly differs from that mediated by the type-1 InsP3 receptor by its lack of stimulation by sulfhydryl oxidation and its lower ATP and InsP3 sensitivity. The predominant expression of the type-3 InsP3 receptor in the bronchial mucosa may be part of a mechanism coping with oxidative stress in that tissue.

Adenosine↗

PubMed1998

Evidence for the intracellular location of chloride channel (ClC)-type proteins: co-localization of ClC-6a and ClC-6c with the sarco/endoplasmic-reticulum Ca2+ pump SERCA2b.

Chloride channel protein (ClC)-6a and ClC-6c, a kidney-specific splice variant with a truncated C-terminus, are proteins that belong structurally to the family of voltage-dependent chloride channels. Attempts to characterize functionally ClC-6a or ClC-6c in Xenopus oocytes have so far been negative. Similarly, expression of both ClC-6 isoforms in mammalian cells failed to provide functional information. One possible explanation of these negative results is that ClC-6 is an intracellular chloride channel rather than being located in the plasma membrane. We therefore studied the subcellular location of ClC-6 isoforms by transiently transfecting COS and CHO cells with epitope-tagged versions of ClC-6a and ClC-6c. Confocal imaging of transfected cells revealed for both ClC-6 isoforms an intracellular distribution pattern that clearly differed from the peripheral location of CD2, a plasma-membrane glycoprotein. Furthermore, dual-labelling experiments of COS cells co-transfected with ClC-6a or -6c and the sarco/endoplasmic-reticulum Ca2+ pump (SERCA2b) indicated that the ClC-6 isoforms co-localized with the SERCA2b Ca2+ pump. Thus ClC-6a and ClC-6c are intracellular membrane proteins, most likely residing in the endoplasmic reticulum. In view of their structural similarity to proven chloride channels, ClC-6 isoforms are molecular candidates for intracellular chloride channels.

Animals↗

PubMed1998

Inhibition of inositol trisphosphate-induced calcium release by cyclic ADP-ribose in A7r5 smooth-muscle cells and in 16HBE14o- bronchial mucosal cells.

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 microM, and in permeabilized 16HBE14o- bronchial mucosal cells with an IC50 of 35 microM. 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 microM) 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.

Adenine Nucleotides↗

PubMed1998

ATP-induced Ca2+ signals in bronchial epithelial cells.

Ca2+-dependent Cl- secretion in the respiratory tract occurs physiologically or under pathophysiological conditions when inflammatory mediators are released. The mechanism of intracellular Ca2+ release was investigated in the immortalized bronchial epithelial cell line 16HBE14o-. Experiments on both intact and permeabilized cells revealed that only inositol 1,4,5-trisphosphate (InsP3) receptors and not ryanodine receptors are involved in intracellular Ca2+ release. The expression pattern of the three InsP3 receptor isoforms was assessed both at the mRNA and at the protein level. The level of expression at the mRNA level was type 3 (92.5%) >> type 2 (5.4%) > type 1 (2.1%) and this rank order was also observed at the protein level. The ATP-induced Ca2+ signals in the intact cell, consisting of abortive Ca2+ spikes or fully developed [Ca2+] rises and intracellular Ca2+ waves, were indicative of positive feedback of Ca2+ on the InsP3 receptors. Low Ca2+ concentrations stimulated and high Ca2+ concentrations inhibited InsP3-induced Ca2+ release in permeabilized 16HBE14o- cells. We localized a cytosolic Ca2+-binding site between amino acid residues 2077 and 2101 in the type-2 InsP3 receptor and between amino acids 2030 and 2050 in the type-3 InsP3 receptor by expressing the respective parts of these receptors as glutathione S-transferase fusion proteins in bacteria. We conclude that the InsP3 receptor isoforms expressed in 16HBE14o- cells (mainly type-3 and type-2) are stimulated by Ca2+ and that this phenomenon contributes to the ATP-induced Ca2+ signals in intact 16HBE14o- cells.

Adenosine Triphosphate↗

PubMed1998

Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-trisphosphate receptors in A7r5 and DDT1 MF-2 smooth muscle cells.

Prolonged stimulation of rat A7r5 aortic smooth muscle cells with 3 microM vasopressin, or of hamster DDT1 MF-2 smooth muscle cells with 10 microM bradykinin or 100 microM histamine led within 4 h to a 40-50% down-regulation of the type 1 InsP3 receptor (InsP3R-1) and of the type 3 InsP3 receptor (InsP3R-3). InsP3R down-regulation was a cell- and agonist-specific process, since several other agonists acting on PLC-coupled receptors did not change the expression level of the InsP3R isoforms in these cell types and since no agonist-induced down-regulation of InsP3Rs was observed in HeLa cells. Down-regulation of InsP3Rs was prevented by an inhibitor of proteasomal protease activity, N-acetyl-Leu-Leu-norleucinal (ALLN). The Ca2+ channel blocker verapamil (2 microM) also induced InsP3R-1 down-regulation (43%) in A7r5 cells, which was inhibited by ALLN. In A7r5 cells transiently transfected with a cDNA construct, bearing a luciferase coding sequence under control of the rat InsP3R-1 promoter, reduced luciferase activity could be demonstrated upon stimulation of cells with vasopressin or verapamil. Thus, besides enhanced protein degradation, a reduction of InsP3R promoter activity might contribute to the down-regulation of InsP3Rs in A7r5 cells. We next investigated the effect of InsP3R down-regulation on Ca2+ responses in A7r5 cells. A rightward shift in the dose-response curve for InsP3-induced Ca2+ release was observed in permeabilized monolayers of vasopressin-pretreated A7r5 cells (EC50 630 nM and 400 nM for pretreated and non-pretreated cells, respectively). The Ca2+ responses to threshold doses of vasopressin were markedly reduced in intact vasopressin-pretreated cells. We conclude that prolonged agonist-exposure leads to down-regulation of InsP3Rs in A7r5 and DDT, MF-2 smooth muscle cells. The mechanism of down-regulation likely involves proteasomal degradation and reduction of InsP3R promoter activity. Moreover, down-regulation of InsP3Rs resulted in desensitization of Ca2+ release from InsP3 sensitive stores.

Adenosine Triphosphate↗

PubMed1998

Changes in actin cytoskeleton during volume regulation in C6 glial cells.

Changes in actin cytoskeleton in the C6 rat glial cell line were studied during decrease or increase (abrupt or gradual) of extracellular osmolality. Actin cytoskeleton was visualized by confocal microscopy after FITC-phalloidin labeling. G-actin, Triton-soluble F-actin and Triton-insoluble F-actin subfractions were determined by gel electrophoresis and scanning, and by DNase I inhibition assays. In control conditions C6 glial cells exhibited well-defined stress fibers and a relatively smooth cortical network. Extracellular anisosmotic changes induced a rapid actin cytoskeletal reorganization, which further progressed and was not reversed upon cell volume recovery. Hypotonic shock caused membrane ruffling and a shift towards polymerized actin, whereas hypertonicity (abrupt or gradual) led to a distinct morphological appearance of abundant short actin microfilaments with, however, no detectable alteration in actin subfractions. When anisosmotic cell volume regulation was prevented, cytoskeleton reorganization depended on the osmotic change and the experimental protocol, but was not related to the absence of volume readjustment. Therefore, although involvement of cytoskeletal alterations in transduction of volume regulatory responses cannot be excluded, it is likely that the observed changes in actin cytoskeleton in C6 glial cells are linked with, but do not initiate, cell volume regulatory processes.

Actins↗

PubMed1997

Molecular and functional evidence for multiple Ca2+-binding domains in the type 1 inositol 1,4,5-trisphosphate receptor.

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+.

Amino Acid Sequence↗

PubMed1997

Effect of a cytosolic Ca2+ concentration ramp on InsP3-induced Ca2+ release in A7r5 smooth-muscle cells and in EBTr cells from tracheal mucosa.

Inositol trisphosphate (InsP3) mediated Ca2+ release is modulated in a complex way by Ca2+. It is not known how the InsP3 receptor responds to a slowly increasing cytosolic [Ca2+]. Two different cell lines (A7r5 smooth-muscle cells and EBTr cells from tracheal mucosa) were investigated. We have now stimulated the Ca2+ stores of the permeabilized cells with a near-threshold [InsP3] and then increased the cytosolic [Ca2+] from 5 nM to 3 microM in 55 steps each lasting 6 s. The rate of InsP3-induced Ca2+ release abruptly increased around 100 nM Ca2+ and reached a maximum at 300 nM Ca2+, above which the Ca2+ release became smaller. The stimulatory effect of cytosolic Ca2+ was much less than that induced by elevating the [InsP3]. The time course of activation by Ca2+ in permeabilized cells resembles the fast InsP3-induced Ca2+ release following the pacemaker [Ca2+] rise in the agonist-stimulated intact cell.

Animals↗

PubMed1997

Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release.

The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1, 4,5)P3 receptor. It was observed that 3'-phosphoadenosine 5'-phosphoulphate, CoA, di(adenosine-5')tetraphosphate (Ap4A) and di(adenosine-5')pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4, 5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.

Adenine Nucleotides↗

PubMed1997

Isoprenylated human brain type I inositol 1,4,5-trisphosphate 5-phosphatase controls Ca2+ oscillations induced by ATP in Chinese hamster ovary cells.

D-myo-Inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase and 3-kinase are thought to be critical regulatory enzymes in the control of InsP3 and Ca2+ signaling. In brain and many other cells, type I InsP3 5-phosphatase is the major phosphatase that dephosphorylates InsP3 and D-myo-inositol 1,3,4,5-tetrakisphosphate. The type I 5-phosphatase appears to be associated with the particulate fraction of cell homogenates. Molecular cloning of the human brain enzyme identifies a C-terminal farnesylation site CVVQ. Post-translational modification of this enzyme promotes membrane interactions and changes in specific activity. We have now compared the cytosolic Ca2+ ([Ca2+]i) responses induced by ATP, thapsigargin, and ionomycin in Chinese hamster ovary (CHO-K1) cells transfected with the intact InsP3 5-phosphatase and with a mutant in which the C-terminal cysteine cannot be farnesylated. [Ca2+]i was also measured in cells transfected with an InsP3 3-kinase construct encoding the A isoform. The Ca2+ oscillations detected in the presence of 1 microM ATP in control cells were totally lost in 87.5% of intact (farnesylated) InsP3 5-phosphatase-transfected cells, while such a loss occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells. All cells overexpressing the InsP3 3-kinase also responded with an oscillatory pattern. However, in contrast to control cells, the [Ca2+]i returned to base-line levels in between a couple of oscillations. The [Ca2+]i responses to thapsigargin and ionomycin were identical for all cells. The four cell clones compared in this study also behaved similarly with respect to capacitative Ca2+ entry. In permeabilized cells, no differences in extent of InsP3-induced Ca2+ release nor in the threshold for InsP3 action were observed among the four clones and no differences in the expression levels of the various InsP3 receptor isoforms could be shown between the clones. Our data support the contention that the ATP-induced increase in InsP3 concentration in transfected CHO-K1 cells is essentially restricted to the site of its production near the plasma membrane, where it can be metabolized by the type I InsP3 5-phosphatase. This enzyme directly controls the [Ca2+]i response and the Ca2+ oscillations in intact cells.

Adenosine Triphosphate↗