Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes.

In hepatocytes, as in other cell types, Ca(2+) signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP(3)R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca(2+) signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP(3) receptors (InsP(3)R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP(3)R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP(3)-induced Ca(2+) release in hepatocytes. This can be explained by the rather low expression level expression of InsP(3)R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca(2+) signaling via an inhibitory effect on InsP(3) synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP(3) synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.

Developmental expression of the calcium release channels during early neurogenesis of the mouse cerebral cortex.

The developmental changes of intracellular calcium release channels of mouse neocortex were studied at the onset of neurogenesis, which occurs between embryonic days E11 and E17. The three main isoforms of the two families of intracellular calcium release channels, namely the inositol trisphosphate receptors (IP3R) and the ryanodine receptors (RyR), were detected by their transcripts in the cerebral hemispheres, as early as stage E11. The major isoforms of each family, IP3R-1 and RyR-2, were found at the protein level by Western blot analysis. Expression of these proteins increases progressively throughout brain development. Their localization in coronal sections of cortex has been observed by immunodetection from E12, and compared to the TuJ1 (anti-class III beta-tubulin antibody) neuronal specific labelling. The expression of both channels is greatly enhanced after E12, and both were seen to be present in most of the proliferative and neuronal cells of the slice. Between E12 and E13, there is a striking transition in the pattern of calcium release elicited by specific agonists of these channels, thimerosal for IP3R and caffeine for RyR. The signals induced by thimerosal were not zone-specific, while the observed calcium release signals induced by caffeine were predominantly restricted out of the ventricular zone. This zone-specific caffeine sensitivity is consistent with the main RyR localization immunodetected at E13. Our results indicate that there is a time lag of several days between the molecular detection of calcium release channels and their functional expression, around the time of neuronal differentiation. Altogether, they provide a molecular basis for analyzing the developmental modulation of calcium signals useful for neurogenesis progression.

Expression of intracellular calcium channels and pumps after partial hepatectomy in rat.

Ca(2+) signals regulate many cellular functions, including proliferation. They are governed by the inositol 1,4,5-trisphosphate receptor (IP(3)R), the only intracellular hepatic Ca(2+) channel and by the endoplasmic reticulum Ca(2+) pumps, SERCA. To characterise their role in regeneration, expression of their isoforms was studied after 2/3 hepatectomy by real-time quantitative PCR, Western blot and binding studies. We found an early increase in the expression of the IP(3)R isoform 1 which contrasted with the decrease of the expression of the IP(3)R isoforms 2 and 3 and of SERCA3. This results in a transient switch between IP(3)R isoforms 1 and 2, IP(3)R isoform 1 becoming predominant before the first round of mitosis. Binding studies detected a 30% diminution of the IP(3)R population at 24 h. In conclusion, the Ca(2+) signalling machinery is regulated, after hepatectomy, by changes in expression of the IP(3)R and SERCA isoforms to adapt Ca(2+) signals to the regenerative state.

Differential expression of inositol 1,4,5-trisphosphate receptor types 1, 2, and 3 in rat myometrium and endometrium during gestation.

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF(4)(-) was enhanced with advancing gestation. (3)[H]IP(3) binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP(3)R as well as their corresponding proteins, IP(3)R-1, IP(3)R-2, and IP(3)R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP(3)Rs increased with advancing gestation, except for IP(3)R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP(3)Rs. The difference noted between the two uterine tissues suggests that IP(3)Rs may have cell-specific functions.

Ligand-binding affinity of the type 1 and 2 inositol 1,4,5-trisphosphate receptors: effect of the membrane environment.

The inositol 1,4,5-trisphosphate (InsP3) receptor is essential for Ca2+ release from intracellular stores. There are three InsP3 receptor types which are targets for several types of regulation. Ca2+, phosphorylation, and protein-protein interactions may contribute to the complex pattern of the Ca2+ signal in stimulated cells. Furthermore, the 3 receptor types could have different affinities for InsP3. We compared the affinities of the type 1 receptor from the cerebellum with the liver type 2 receptor both in their membrane environment and after isolation by immunoprecipitation. Measurements of [3H]InsP3 binding in a cytosol-like medium revealed that the Kd of the liver receptor (45 +/- 5 nM, N = 14) was higher than the Kd of the cerebellar receptor (28 +/- 3 nM, N = 9). Solubilization and immunopurification of the liver InsP3 receptor resulted in a 10-fold increase in its affinity for InsP3. The affinity of the cerebellar receptor did not change under these conditions. Therefore, the extraction of the liver and the cerebellar receptors from their membrane environments induced an inversion of their relative affinities. Treatment of liver membranes with low concentrations of detergents also increased the affinity for InsP3 binding. These data indicate that the type 1 and the type 2 InsP3 receptors have different affinities for InsP3 and that the properties of the type 2 receptor are strongly regulated by hydrophobic interactions within its membrane environment.

The properties of a subtype of the inositol 1,4,5-trisphosphate receptor resulting from alternative splicing of the mRNA in the ligand-binding domain.

Subtypes of the type-1 inositol 1,4,5-trisphosphate (InsP3) receptor differ at the mRNA level in two small variably spliced segments. One segment (SI) encodes for a sequence within the InsP3-binding domain, thus its presence or absence could affect the functions of the receptor. We have used anti-peptide antibodies to confirm the existence of different subtypes of the InsP3 receptor (InsP3R) protein. The antibody against residues 322-332, within the SI region, recognized a 260 kDa polypeptide in membranes prepared from rat cerebellum or cerebral cortex. The cerebellum contained a few percent of the InsP3R protein having the SI region, whereas the cerebral cortex contained a high proportion of receptors with the SI region. These two tissues were representative of both isoforms, SI- or SI+, and displayed the same [3H]InsP3-binding characteristics. Thus, the SI region was not involved in the basic properties of the receptor. Deletion of the peptide 316-352 containing the SI segment greatly reduced InsP3 binding [Miyawaki, Furuichi, Ryou, Yoshikawa, Nakagawa, Saitoh and Mikoshiba (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4911-4915]. The antibodies against the SI region or against residues 337-349 did not modify the binding of [3H]InsP3 in the cortical membranes rich in the SI+ isoform or in cerebellar membranes. These results suggested that the SI region was not part of the binding site. The subcellular distribution of these two isoforms was then investigated in rat liver. The two isoforms were identified in different membrane fractions and they followed the same subcellular distribution. We suggest that the domain with the SI region may be involved in a function other than InsP3-induced Ca2+ release.

The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver.

Inositol 1,4,5-trisphosphate (InsP3) is involved in the mobilization of Ca2+ from intracellular non-mitochondrial stores. In rat liver, it has been shown that the InsP3-binding site co-purifies with the plasma membrane. This suggests that in the liver the InsP3 receptor (InsP3R) associates with plasma membrane. We studied the subcellular distribution of the liver InsP3R by measuring the maximal binding capacity of [3H]InsP3 and using antibodies against the 14 C-terminal residues of the type 1 InsP3R. The antibodies recognized a large amount of an InsP3R protein of 260 kDa in a membrane fraction which is also enriched with [3H]InsP3-binding sites and with markers of the basal, the lateral and the bile-canalicular membrane and the plasma-membrane Ca2+ pump (PMCA). The fractions enriched in markers of the endoplasmic reticulum (ER) and the Ca2+ pump of the ER (SERCA2b) contained low levels of InsP3 receptors. The immunofluorescent labelling of cultured hepatocytes with anti-InsP3R antibodies indicated that the receptor is concentrated in the perinuclear area and in some regions near the plasma membrane. The fraction enriched with InsP3R is also contaminated with markers of the ER and with SERCA2b. It was exposed to alkaline medium (pH 10.5) to extract endogenous actin and membrane-associated proteins before being subfractionated by Percoll-gradient centrifugation. The alkaline treatment allowed partial separation of the markers of the ER from the markers of the plasma membrane. The InsP3R was recovered in the heavy subfraction, which was also enriched with markers for the ER and with the SERCA2b and contained low levels of markers of the plasma membrane. These data indicate that the InsP3R is neither localized on the plasma membrane itself nor homogeneously distributed on the ER membrane. This supports the view that part of the receptor is localized on a specialized sub-region of the ER which interacts with the plasma membrane.

The inositol 1,4,5-trisphosphate receptor: kinetic properties and regulation.

Inositol 1,4,5-triphosphate (InsP3) is a second messenger responsible for the mobilization of intracellular Ca2+ after receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. InsP3 binds to a specific receptor located on the membrane of an intracellular compartment and opens a Ca2+ channel causing the cytosolic Ca2+ concentration to increase. Measurement of radiolabelled InsP3 binding and InsP3-induced Ca2+ release in parallel experiments indicated that the liver InsP3 receptor exists in two main states: an active state (A) and an inactive one (I). The "I" form of the receptor is found in the presence of high Ca2+ concentrations (above 1 microM). The binding properties of the "A" and the "I" states of the receptor have been characterized by analysing a membrane fraction enriched in InsP3 receptors. The inactive "I" state displays a high affinity (Kd = 2 nM) and slow rates of association and dissociation. The active state "A" of the receptor displays complex kinetic properties. The rate of association and the rate of dissociation of labelled InsP3 are rapid phenomena probably involving several components. The apparent Kd for the InsP3 binding is about 40 nM in a low Ca2+ medium. The affinity of the "A" state of the receptor is increased by Ca2+ (at concentrations lower than 0.5 microM) and by thiol reagents. The increase of the affinity of the receptor is due to a decrease of the dissociation rate constants. This lowers the threshold such that Ca2+ is released at lower concentrations of InsP3. These data indicate that the binding of InsP3 to its receptor is a complex phenomenon involving the transition among several states.(ABSTRACT TRUNCATED AT 250 WORDS)

Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor.

Thiol reagents have been shown to increase cytosolic Ca2+ in several cell types. In non-muscle cells, these agents induce Ca2+ spikes by increasing the sensitivity of the intracellular Ca2+ stores to D-myo-inositol-1,4,5-trisphosphate (InsP3). We have investigated the effects of thimerosal and oxidized glutathione on the binding properties of the InsP3 receptor in permeabilized hepatocytes and liver and cerebellar membranes. Thimerosal, at the maximal concentration of 100 microM, decreased the KD for the InsP3 binding to permeabilized hepatocytes and cerebellar membranes from 16 to 3 nM and from 25 to 8 nM, respectively, without affecting the maximal binding capacities. On liver membranes, both thimerosal and high Ca2+ concentrations increased the affinity for InsP3 binding. The Ca2+ and the thimerosal effects were differentiated by kinetic experiments. In low Ca2+ media, two kinetic components were identified and thimerosal decreased the rate of dissociation from both these components without affecting the rate of association. In the high Ca2+ medium, a single kinetic component was found with a very slow rate of dissociation. These data suggest that the InsP3 receptor exists in different states. The high-affinity inactive state induced by high Ca2+ concentrations displays slow rates of association and dissociation. The binding properties of the receptor in its active state can be regulated by thiol reagents which increase the affinity by decreasing the dissociation rate constants. At a resting concentration of 100-200 nM, Ca2+ has two effects: it increases the affinity of the active state of the receptor as thiol reagents do and transforms part of the receptors into the inactive high-affinity state.

Characterization of a rapidly dissociating inositol 1,4,5-trisphosphate-binding site in liver membranes.

The binding of inositol 1,4,5-trisphosphate (InsP3) to a specific receptor induces the release of Ca2+ from an intracellular store. In the liver, the KD of a low affinity state of the receptor (RL) found at low Ca2+ concentration ([Ca2+]) is in close agreement with the EC50 of the InsP3-induced Ca2+ release. We have developed an experimental procedure for measuring the rate of dissociation of this low affinity [32P]InsP3-receptor complex in less than 1 s. When the receptor was in the RL state, two kinetic components, RL1 and RL2, were identified with respective rate constants (k(off)) of 1-2 s-1 and 0.03-0.06 s-1. Increasing the [Ca2+] up to 1 microM transformed the receptor into the high affinity state (RH) and decreased the dissociation rate constant to 2 x 10(-2) min-1. We also investigated the time course of the transformation of the receptor from the high affinity (RH) to the low affinity state (RL) after decreasing the [Ca2+] to less than 10 nM. This reversion was dramatically dependent on temperature: at 4 degrees C, the receptor was locked in the RH state, whereas at 37 degrees C the receptor reverted to the RL state with a half-time of less than 1 s. The reversion from the RH state to the RL one is associated to a recovery of InsP3-induced 45Ca2+ release on permeabilized hepatocytes. The rapid and reversible transformation of the InsP3 receptor from an active to an inactive state may be a key event in the Ca2+ release process in intact cells.

Decreased G3PDH binding to erythrocyte membranes in sickle cell disease.

Several membrane abnormalities have been described in red cells from patients with sickle cell disease, responsible for chronic hemolytic anemia. We describe here a 35-50% inhibition of the binding of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) to the membrane of sickle red cells. Varying the phosphorylation state of the membrane proteins did not change their affinity for the enzyme. Protein band 3 and the cytoplasmic domain of this protein isolated from sickle red cells showed normal interaction with the enzyme. The inhibition observed with intact membranes is not due to short term oxidation of membrane proteins, as various procedures inducing acute oxidative stress in normal membranes did not reproduce the inhibition of G3PDH binding. We conclude that the alteration of the binding of G3PDH to the membrane of sickle erythrocytes is probably related to long term processes involving cycles of HbS polymer formation.

Calcium mediates the interconversion between two states of the liver inositol 1,4,5-trisphosphate receptor.

D-myo-Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) regulates intracellular Ca2+ by mobilizing Ca2+ from a non-mitochondrial store. We have investigated the effects of Ca2+ on the binding of [32P]Ins (1,4,5)P3 to permeabilized rat hepatocytes and a liver plasma membrane-enriched fraction. Increasing the free Ca2+ concentration in the medium from 0.1 nM to 0.7 microM increased the capacity of a high affinity binding component (KD = 2-3 nM) in permeabilized cells by a factor of 10. If the membrane fraction was preincubated at 37 degrees C before binding was measured at 4 degrees C, all of the Ins(1,4,5)P3 receptors were transformed to a low affinity state (KD = 65 +/- 12 nM, Bmax = 3.1 +/- 0.1 fmol/mg, n = 4). When 0.7 microM of Ca2+ was added, the receptors were totally transformed to a high affinity state (KD = 2.8 +/- 0.4 nM, Bmax = 2.7 +/- 0.4 fmol/mg, n = 4). The EC50 of the Ca2(+)-induced interconversion of the Ins(1,4,5)P3 receptor was 140 nM. This Ca2(+)-induced transformation of the Ins(1,4,5)P3 receptor from a low affinity to a high affinity state was associated with an inhibition of the Ins(1,4,5)P3-induced Ca2+ release in permeabilized hepatocytes. These data suggest that the Ins(1,4,5)P3-dependent hormones, by increasing the intracellular Ca2+ concentration, induce a reversible transformation of the receptor from its low affinity state, coupled to the Ca2+ release, to a desensitized high affinity state. Transformation of the receptor may play a role in the oscillatory release of Ca2+ observed in single isolated hepatocytes.

[Characterisation of 2 reversible states of the inositol 1,4,5-trisphosphate receptor in rat hepatocytes]. / Caractérisation de deux états interconvertibles du récepteur de l'inositol 1,4,5-trisphosphate dans les hépatocytes de rat.

Myo-inositol-1,4,5-trisphosphate or (I(1,4,5)P3) is generated in liver cells after hormonal stimulation. It bind to specific receptors and induces the release of Ca2+ from an intracellular store. This receptor has been found in permeabilized hepatocytes and showed two states of low- and high-affinity with KD of 1-2 and 40-50 nmol/l, respectively. Measurements of 45Ca2+ release, mediated by different analogues, revealed that the low-affinity site was coupled to the Ca2+ channel open state. The pretreatment of cells with vasopressin, an I(1,4,5)P3-dependent agonist, induced an 60 percent increase of the binding capacity of the high-affinity sites. Incubation with 1 mumol/l Ca2+ increased the number of high-affinity sites from 5 to 65 fmol/10(6) cells. This effect was associated with a decrease in the number of low-affinity sites from 130 to 80 fmol/10(6) cells. Our results suggest that the intracellular Ca2+ concentration rise mediated by I(1,4,5)P3-dependent agonists, induced a conversion of the low-affinity form of the I(1,4,5)P3 receptor, coupled to Ca2+ release, to a desensitized high-affinity one. This process could explain the oscillations of the intracellular Ca2+ concentration observed in hormone-treated single cells.

Characterization of two forms of inositol 1,4,5-trisphosphate receptor in rat liver.

Two binding sites for [32P]myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) were detected in a crude particulate fraction prepared from rat liver homogenate and in permeabilized hepatocytes. The same high- and low-affinity sites with KDs of 1.8-2.6 nM and 35-71 nM, respectively, were detected in subcellular fractions enriched in plasma membranes, mitochondria and microsomes, with relative proportions close to those found in the crude membrane fraction. The order of potency of three inositol phosphates in inhibiting [32P]Ins(1,4,5)P3 binding to the two sites, i.e. Ins(1,4,5)P3 greater than Ins(2,4,5)P3] greater than Ins(1,3,4,5)P4, and the inhibition by heparin, strongly suggest that neither of the binding sites reflected components due to the 3-kinase or the 5-phosphatase. A close correlation was observed between the dose-response curves for Ca2+ release by Ins(1,4,5)P3 and Ins(2,4,5)P3 and the occupancy of the low-affinity binding site by these agonists. These results support the view that the two [32P]Ins(1,4,5)P3 binding sites are two forms of the same receptor.

Hormonal regulation of inositol 1,4,5-trisphosphate receptor in rat liver.

Inositol 1,4,5-trisphosphate (IP3) is a second messenger which induces Ca2+ release from an intracellular store. We have investigated the properties of the [32P]IP3 binding sites in rat liver. Two specific [32P]IP3 receptors with KD of 2.3 and 88 nM and respective capacities of 33 fmol/mg protein and 195 fmol/mg protein have been detected in a crude membrane fraction prepared from rat liver homogenate. The pretreatment of the liver with IP3-dependent hormones increased two-fold the capacity of the high affinity site. This effect was partly reversed by dibutyryl cyclic AMP. Permeabilized hepatocytes also displayed two [32P]IP3 binding sites with KD of 1.5 and 84 nM and respective capacities of 8 and 300 fmol/10(6) cells. We have measured the [32P]IP3 binding and the IP3-induced 45Ca2+ release in the same batch of permeabilized hepatocytes. In a low Mg2+ medium, the EC50 for 45Ca2+ release was in close correlation with the KD for the low affinity site. These data suggest that an equilibrium between two states of the IP3 receptor is regulated by hormone action and the low affinity state is responsible for the intracellular Ca2+ release.

Characterization of Ca2+ fluxes in rat liver plasma-membrane vesicles.

Inside-out plasma-membrane vesicles isolated from rat liver [Prpic, Green, Blackmore & Exton (1984) J. Biol. Chem. 259, 1382-1385] accumulated a substantial amount of 45Ca2+ when they were incubated in a medium whose ionic composition and pH mimicked those of cytosol and which contained MgATP. The Vmax of the initial 45Ca2+ uptake rate was 2.9 +/- 0.6 nmol/min per mg and the Km for Ca2+ was 0.50 +/- 0.08 microM. The ATP-dependent 45Ca2+ uptake by inside-out plasma-membrane vesicles was about 20 times more sensitive to saponin than was the ATP-dependent uptake by a microsomal preparation. The 45Ca2+ efflux from the inside-out vesicles, which is equivalent to the Ca2+ influx in intact cells, was increased when the free Ca2+ concentration in the medium was decreased. The Ca2+ antagonists La3+ and Co2+ inhibited the 45Ca2+ efflux from the vesicles. Neomycin stimulated the Ca2+ efflux in the presence of either a high or a low free Ca2+ concentration. These results confirm that polyvalent cations regulate Ca2+ fluxes through the plasma membrane.

The influence of nitroheterocyclic radiosensitizers on the membrane of red blood cells.

Nitroimidazole compounds are effective radiosensitizers, but neurotoxic side effects prevent their clinical use. Studying the effect of misonidazole, metronidazole and two of its derivatives, 4.5-NO2-METRO and 4-NO2-METRO, on red blood cell, it was recently demonstrated that these compounds inhibit the red cell membrane (Na+-K+) ATPase and decrease the fluidity of the membrane bilayer. In order to extend these observations and to achieve a more complete interpretation, four additional investigations were selected: the (Ca++-Mg++)ATPase activity, the anion channel (band 3 protein) kinetics, the susceptibility of the phospholipids to peroxidation, and their influence on the concentration of reduced glutathione (GSH). The activity of the (Ca++-Mg++)ATPase and its stimulation by calmodulin were decreased by all four drugs, but the anion transport kinetics were unaltered. No lipid peroxidation could be detected, as estimated by the production of malonyldialdehyde. The red cell GSH was depleted by 4.5-NO2-METRO, probably due to the formation of a complex between GSH and the drugs [Varghese 1983]. The mechanism of the inhibition of the ATPases is not yet clearly apparent; it is presently sought in a direct interaction of the drugs with some thiol reactive groups of the ATPases.

Organic phosphates modulate anion self-exchange across the human erythrocyte membrane.

Anion transport across the red cell membrane has been measured as sulfate self-exchange flux (Ja) in fresh and metabolically depleted human red cells. Depletion of metabolic stores by a starvation of the cells decreases Ja by 50%. A similar effect was observed when ATP was acutely and selectively depleted by iodoacetamide. This inhibition was independent of the presence of calcium and reversible after metabolic rejuvenation of the cells. Ghosts prepared from fresh red cells exhibited the same value of Ja as fresh red cells. By contrast, ghosts prepared from depleted red cells exhibited a decrease in Ja which was reverted by a physiological concentration of ATP. The effect of ATP was dependent on its concentration (Km approximately 40 microM) and on the duration of the metabolic depletion: complete restoration of Ja was obtained only in ghosts prepared from red cells acutely depleted of ATP by a 2 h incubation with iodoacetamide. After a 20 h starvation, Ja restoration was never more than 80%. We postulate that ATP acts primarily through the phosphorylation of band 3 protein, the anion exchanger; it acts also through the stabilization of the normal organization of the membrane. This latter effect may involve the phosphorylation of membrane components, but also a direct interaction, as shown by the influence of other organic phosphates (2,3-diphosphoglycerate and inositol hexaphosphate) on Ja in the absence of ATP.