Enhanced caffeine-induced Ca2+ release in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly and is a complex disorder that involves altered proteolysis, oxidative stress and disruption of ion homeostasis. Animal models have proven useful in studying the impact of mutant AD-related genes on other cellular signaling pathways, such as Ca2+ signaling. Along these lines, disturbances of intracellular Ca2+ ([Ca2+]i) homeostasis are an early event in the pathogenesis of AD. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate disturbances in Ca2+ homeostasis in primary cortical neurons from a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). Application of caffeine to mutant presenilin-1 knock-in neurons (PS1KI) and 3xTg-AD neurons evoked a peak rise of [Ca2+]i that was significantly greater than those observed in non-transgenic neurons, although all groups had similar decay rates of their Ca2+ transient. This finding suggests that Ca2+ stores are greater in both PS1KI and 3xTg-AD neurons as calculated by the integral of the caffeine-induced Ca2+ transient signal. Western blot analysis failed to identify changes in the levels of several Ca2+ binding proteins (SERCA-2B, calbindin, calsenilin and calreticulin) implicated in the pathogenesis of AD. However, ryanodine receptor expression in both PS1KI and 3xTg-AD cortex was significantly increased. Our results suggest that the enhanced Ca2+ response to caffeine observed in both PS1KI and 3xTg-AD neurons may not be attributable to an alteration of endoplasmic reticulum store size, but to the increased steady-state levels of the ryanodine receptor.     

Dysregulated ryanodine receptors mediate cellular toxicity: restoration of normal phenotype by FKBP12.6

Ca2+ homeostasis is a vital cellular control mechanism in which Ca2+ release from intracellular stores plays a central role. Ryanodine receptor (RyR)-mediated Ca2+ release is a key modulator of Ca2+ homeostasis, and the defective regulation of RyR is pathogenic. However, the molecular events underlying RyR-mediated pathology remain undefined. Cells stably expressing recombinant human RyR2 (Chinese hamster ovary cells, CHOhRyR2) had similar resting cytoplasmic Ca2+ levels ([Ca2+]c) to wild-type CHO cells (CHOWT) but exhibited increased cytoplasmic Ca2+ flux associated with decreased cell viability and proliferation. Intracellular Ca2+ flux increased with human RyR2 (hRyR2) expression levels and determined the extent of phenotypic modulation. Co-expression of FKBP12.6, but not FKBP12, or incubation of cells with ryanodine suppressed intracellular Ca2+ flux and restored normal cell viability and proliferation. Restoration of normal phenotype was independent of the status of resting [Ca2+]c or ER Ca2+ load. Heparin inhibition of endogenous inositol trisphosphate receptors (IP3R) had little effect on intracellular Ca2+ handling or viability. However, purinergic stimulation of endogenous IP3R resulted in apoptotic cell death mediated by hRyR2 suggesting functional interaction occurred between IP3R and hRyR2 Ca2+ release channels. These data demonstrate that defective regulation of RyR causes altered cellular phenotype via profound perturbations in intracellular Ca2+ signaling and highlight a key modulatory role of FKBP12.6 in hRyR2 Ca2+ channel function.     

Up-regulation of inositol 1,4,5-trisphosphate receptor type 1 is responsible for a decreased endoplasmic-reticulum Ca2+ content in presenilin double knock-out cells

Presenilins (PS) are proteins involved in the pathogenesis of autosomal-dominant familial cases of Alzheimer's disease. Mutations in PS are known to induce specific alterations in cellular Ca2+ signaling which might be involved in the pathogenesis of neurodegenerative diseases. Mouse embryonic fibroblasts (MEF) deficient in PS1 and PS2 (PS DKO) as well as the latter rescued with PS1 (Rescue), were used to investigate the underlying mechanism of these alterations in Ca2+ signaling. PS DKO cells were characterized by a decrease in the [Ca2+]ER as measured by ER-targeted aequorin luminescence and an increased level of type 1 inositol 1,4,5-trisphosphate receptor (IP3R1). The lower [Ca2+]ER was associated with an increase in a Ca2+ leak from the ER. The increased IP3R1 expression and the concomitant changes in ER Ca2+ handling were reversed in the Rescue cells. Moreover using RNA-interference mediated reduction of IP3R1 we could demonstrate that the up-regulation of this isoform was responsible for the increased Ca2+ leak and the lowered [Ca2+]ER PS DKO cells. Finally, we show that the decreased [Ca2+]ER in PS DKO cells was protective against apoptosis.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Presenilin-2 mutations modulate amplitude and kinetics of inositol 1, 4,5-trisphosphate-mediated calcium signals.

Mutations in the two presenilin genes (PS1, PS2) account for the majority of early-onset familial Alzheimer's disease (FAD) cases. Converging evidence from a variety of experimental systems, including fibroblasts from FAD patients and transgenic animals, indicates that PS1 mutations modulate intracellular calcium signaling pathways. Despite the potential relevance of these changes to the pathogenesis of FAD, a comparable effect for PS2 has not yet been demonstrated experimentally. We examined the effects of wild-type PS2, and both of the identified FAD mutations in PS2, on intracellular calcium signaling in Xenopus oocytes. Inositol 1,4, 5-trisphosphate (IP(3))-evoked calcium signals were significantly potentiated in cells expressing either of the PS2 mutations relative to wild-type PS2-expressing cells and controls. Decay rates of calcium signals were also significantly accelerated in mutant PS2-expressing cells in a manner dependent upon IP(3) concentration. The finding that mutations in both PS1 and PS2 modulate intracellular calcium signaling suggests that these disturbances may represent a common pathogenic mechanism of presenilin-associated FAD.     

Mechanisms of integrin-mediated calcium signaling in MDCK cells: regulation of adhesion by IP3- and store-independent calcium influx.

Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.     

Chronic antidepressants potentiate via sigma-1 receptors the brain-derived neurotrophic factor-induced signaling for glutamate release

Up-regulation of BDNF (brain-derived neurotrophic factor) has been suggested to contribute to the action of antidepressants. However, it is unclear whether chronic treatment with antidepressants may influence acute BDNF signaling in central nervous system neurons. Because BDNF has been shown by us to reinforce excitatory glutamatergic transmission in cultured cortical neurons via the phospholipase-gamma (PLC-gamma)/inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway (Numakawa, T., Yamagishi, S., Adachi, N., Matsumoto, T., Yokomaku, D., Yamada, M., and Hatanaka, H. (2002) J. Biol. Chem. 277, 6520-6529), we examined in this study the possible effects of pretreatment with antidepressants on the BDNF signaling through the PLC-gamma)/IP3/Ca2+ pathway. Furthermore, because the PLC-gamma/IP3/Ca2+ pathway is regulated by sigma-1 receptors (Hayashi, T., and Su, T. P. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 491-496), we examined whether the BDNF signaling is modulated by sigma-1 receptors (Sig-1R). We found that the BDNF-stimulated PLC-gamma activation and the ensued increase in intracellular Ca2+ ([Ca2+]i) were potentiated by pretreatment with imipramine or fluvoxamine, so was the BDNF-induced glutamate release. Furthermore, enhancement of the interaction between PLC-gamma and TrkB (receptor for BDNF) after imipramine pretreatment was observed. Interestingly, BD1047, a potent Sig-1R antagonist, blocked the imipramine-dependent potentiation on the BDNF-induced PLC-gamma activation and glutamate release. In contrast, overexpression of Sig-1R per se, without antidepressant pretreatment, enhances BDNF-induced PLC-gamma activation and glutamate release. These results suggest that antidepressant pretreatment selectively enhance the BDNF signaling on the PLC-gamma/IP3/Ca2+ pathway via Sig-1R, and that Sig-1R plays an important role in BDNF signaling leading to glutamate release.     

Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes.

Following receptor activation in Xenopus oocytes, spiral waves of intracellular Ca2+ release were observed. We have identified key molecular elements in the pathway that give rise to Ca2+ excitability. The patterns of Ca2+ release produced by GTP-gamma-S and by inositol 1,4,5-trisphosphate (IP3) are indistinguishable from receptor-induced Ca2+ patterns. The regenerative Ca2+ activity is critically dependent on the presence of IP3 and on the concentration of intracellular Ca2+, but is independent of extracellular Ca2+. Broad regions of the intracellular milieu can be synchronously excited to initiate Ca2+ waves and produce pulsating foci of Ca2+ release. By testing the temperature dependence of wavefront propagation, we provide evidence for an underlying process limited by diffusion, consistent with the elementary theory of excitable media. We propose a model for intracellular Ca2+ signaling in which wave propagation is controlled by IP3-mediated Ca2+ release from internal stores, but is modulated by the cytoplasmic concentration and diffusion of Ca2+.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

Differential inositol 1,4,5-trisphosphate receptor signaling in a neuronal cell line

Differential intracellular distribution of the three pharmacologically and biophysically distinct types of IP3Rs can lead to different subcellular Ca2+ transients each coupled to discrete intracellular functions. Here, we report the functional localization of differentially distributed IP3 receptor types in the commonly-used hippocampal cell line HT22. The distinct subcellular localization and Ca2+ signaling properties of these receptors determine the potential role of specific IP3 receptor types in cellular function. By utilizing immunochemistry, we conclude that HT22 cells express all three IP3 receptors with types 1 and 3 being expressed predominantly in the endoplasmic reticulum and perinuclear regions and type 2 being expressed predominantly in the nuclear envelope. Optical imaging studies using the Ca2+-sensitive indicator dye fluo-3 show that nuclear IP3 responses have greater amplitude and faster kinetics than cytosolic IP3 responses corresponding to the biophysical characteristics of the differentially distributed receptor types. These results support the hypothesis that differentially distributed IP3R isotypes mediate distinct cellular functions through differential, organelle-specific Ca2+ signaling.     

Measurement of intracellular IP3 during Ca2+ oscillations in mouse eggs with GFP-based FRET probe

Intracellular Ca2+ oscillations in fertilized mammalian eggs, the key signal that stimulates egg activation and early embryonic development, are regulated by inositol 1,4,5-trisphosphate (IP3) signaling pathway. We investigated temporal changes in intracellular IP3 concentration ([IP3]i) in mouse eggs, using a fluorescent probe based on fluorescence resonance energy transfer between two green fluorescent protein variants, during Ca2+ oscillations induced by fertilization or expression of phospholipase Czeta (PLCzeta), an egg-activating sperm factor candidate. Fluorescence measurements suggested the elevation of [IP3]i in fertilized eggs, and the enhancement of PLCzeta-mediated IP3 production by cytoplasmic Ca2+ was observed during Ca2+ oscillations or in response to CaCl2 microinjection. The results supported the view that PLCzeta is the sperm factor to stimulate IP3 pathway, and suggested that high Ca2+ sensitivity of PLCzeta activity and positive feedback from released Ca2+ are important for triggering and maintaining Ca2+ oscillations.     

Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes

Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+-signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+-signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signalling.     

Polycystin 2 interacts with type I inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling

Autosomal dominant polycystic kidney disease, a common cause of renal failure, arises from mutations in either the PKD1 or the PKD2 gene. The precise function of both PKD gene products polycystins (PCs) 1 and 2 remain controversial. PC2 has been localized to numerous cellular compartments, including the endoplasmic reticulum, plasma membrane, and cilia. It is unclear what pools are the most relevant to its physiological function as a putative Ca2+ channel. We employed a Xenopus oocyte Ca2+ imaging system to directly investigate the role of PC2 in inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling. Cytosolic Ca2+ signals were recorded following UV photolysis of caged IP3 in the absence of extracellular Ca2+. We demonstrated that overexpression of PC2, as well as type I IP3 receptor (IP3R), significantly prolonged the half-decay time (t1/2) of IP3-induced Ca2+ transients. However, overexpressing the disease-associated PC2 mutants, the point mutation D511V, and the C-terminally truncated mutation R742X did not alter the t1/2. In addition, we found that D511V overexpression significantly reduced the amplitude of IP3-induced Ca2+ transients. Interestingly, overexpression of the C terminus of PC2 not only significantly reduced the amplitude but also prolonged the t1/2. Co-immunoprecipitation assays indicated that PC2 physically interacts with IP3R through its C terminus. Taken together, our data suggest that PC2 and IP3R functionally interact and modulate intracellular Ca2+ signaling. Therefore, mutations in either PC1 or PC2 could result in the misregulation of intracellular Ca2+ signaling, which in turn could contribute to the pathology of autosomal dominant polycystic kidney disease.     

Divergent responses of ras-transfected and non-ras-transfected human keratinocytes to extracellular calcium.

Raising extracellular calcium (Ca(o)) induces terminal differentiation in cultured epidermal keratinocytes. The introduction of the ras oncogene into keratinocytes results in resistance to Ca(o)-mediated differentiation. To understand the signaling mechanism involved, we examined the Ca(o)-induced formation of inositol triphosphate (IP3) and changes in intracellular Ca2+ (Ca(i)) concentration in non-ras-transfected and ras-transfected HaCaT lines of human keratinocytes. When switched from 0.05- to 1.5-mM Ca(o) medium, the non-ras HaCaT line showed a rapid twofold increase in IP3 formation, whereas the IP3 level in the ras-transfected I-7 line was slightly affected. G-protein-coupled activation of phospholipase was intact in both lines, as evidenced by the generation of similar amounts of IP3 in response to addition of bradykinin or guanosine 5'-[gamma-thio]-triphosphate. Addition of 1.0 mM Ca(o) evoked similar Ca(i) responses in both non-ras- and ras-transfected cells: a transient elevation, followed by a sustained lower plateau. However, the two lines differed in their later responses: after being maintained in 1.0 mM Ca2+ for 24 h, the Ca(i) level was significantly lower in ras-transfected cells than in non-ras-transfected HaCaT cells. The Ca(o)-induced increase in Ca(i) in both lines was inhibited by the Ca2+ entry blocker SK&F 96365 or depolarization in high K+ bathing solution, demonstrating its dependence of calcium influx. The results suggest fundamental differences in the early signal that are generated in response to an increase in Ca(o) in ras-transfected keratinocytes, with the absence of a Ca(o)-induced rise in IP3--a signaling pathway defect that may play a role in the differentiation block the cells exhibit. In addition, the inability of ras-transfected cells to sustain a prolonged Ca(i) plateau may also contribute to their inability to differentiate in response to the Ca(o) signal.     

Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.     

A novel follicle-stimulating hormone-induced G alpha h/phospholipase C-delta1 signaling pathway mediating rat sertoli cell Ca2+-influx

FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mm) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 microm), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 microm), did not. On the other hand, the activation of G alpha h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-delta1 from cytosol to cell membrane and the formation of G alpha h /PLC-delta1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-delta1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-delta1 translocation, formation of G alpha h /PLC-delta1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G alpha h /PLC-delta1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.     

Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353

The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3.     

IP3-independent signalling of OX1 orexin/hypocretin receptors to Ca2+ influx and ERK

OX1 orexin receptors (OX1R) have been shown to activate receptor-operated Ca2+ influx pathways as their primary signalling pathway; however, investigations are hampered by the fact that orexin receptors also couple to phospholipase C, and therewith inositol-1,4,5-trisphosphate (IP3)-dependent Ca2+ release. We have here devised a method to block the latter signalling in order to focus on the mechanism of Ca2+ influx activation by OX1R in recombinant systems. Transient expression of the IP3-metabolising enzymes IP3-3-kinase-A (inositol-1,4,5-trisphosphate-->inositol-1,3,4,5-tetrakisphosphate) and type I IP3-5-phosphatase (inositol-1,4,5-trisphosphate-->inositol-1,4-bisphosphate) almost completely attenuated the OX1R-stimulated IP3 elevation and Ca2+ release from intracellular stores. Upon attenuation of the IP3-dependent signalling, the receptor-operated Ca2+ influx pathway became the only source for Ca2+ elevation, enabling mechanistic studies on the receptor-channel coupling. Attenuation of the IP3 elevation did not affect the OX1R-mediated ERK (extracellular signal-regulated kinase) activation in CHO cells, which supports our previous finding of the major importance of receptor-operated Ca2+ influx for this response.