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PubMed2002

The presenilin 1 deltaE9 mutation gives enhanced basal phospholipase C activity and a resultant increase in intracellular calcium concentrations.

We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-DeltaE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca(2+) concentrations ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-DeltaE9 cells have an enhanced basal PI hydrolysis and [Ca(2+)](i) as compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-DeltaE9-related high basal [Ca(2+)](i) was also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca(2+)](i) in PS1-DeltaE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-DeltaE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca(2+). Ruthenium red only reduced carbachol peak elevations of [Ca(2+)](i) in NT and PS1-WT cells and not in PS1-DeltaE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca(2+)](i) with either ryanodine or the endoplasmic reticulum Ca(2+) ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-beta1 showed that, compared with NT and PS1-WT cells, the PS1-DeltaE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-beta1. Our results suggest that the PS1-DeltaE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca(2+)](i). This may provide a novel mechanism by which the PS1-DeltaE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid beta generation.

Amyloid beta-Peptides↗

PubMed2002

Toxoplasma gondii microneme secretion involves intracellular Ca(2+) release from inositol 1,4,5-triphosphate (IP(3))/ryanodine-sensitive stores.

Calcium-mediated microneme secretion in Toxoplasma gondii is stimulated by contact with host cells, resulting in the discharge of adhesins that mediate attachment. The intracellular source of calcium and the signaling pathway(s) triggering release have not been characterized, prompting our search for mediators of calcium signaling and microneme secretion in T. gondii. We identified two stimuli of microneme secretion, ryanodine and caffeine, which enhanced release of calcium from parasite intracellular stores. Ethanol, a previously characterized trigger of microneme secretion, stimulated an increase in parasite inositol 1,4,5-triphosphate, implying that this second messenger may mediate intracellular calcium release. Consistent with this observation, xestospongin C, an inositol 1,4,5-triphosphate receptor antagonist, inhibited microneme secretion and blocked parasite attachment and invasion of host cells. Collectively, these results suggest that T. gondii possess an intracellular calcium release channel with properties of the inositol 1,4,5-triphosphate/ryanodine receptor superfamily. Intracellular calcium channels, previously studied almost exclusively in multicellular animals, appear to also be critical to the control of parasite calcium during the initial steps of host cell entry.

Animals↗

PubMed2004

In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms.

We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3.4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3.4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3.4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. The major BILN 2061-resistant mutations at Asp(168) are fully susceptible to VX-950, and the dominant resistant mutation against VX-950 at Ala(156) remains sensitive to BILN 2061. Modeling analysis suggests that there are different mechanisms of resistance to VX-950 and BILN 2061.

Amino Acids↗

PubMed2004

Polycystin-1 activates the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway.

Regulation of intracellular Ca(2+) mobilization has been associated with the functions of polycystin-1 (PC1) and polycystin-2 (PC2), the protein products of the PKD1 and PKD2 genes. We have now demonstrated that PC1 can activate the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway through Galpha(q) -mediated activation of phospholipase C (PLC). Transient transfection of HEK293T cells with an NFAT promoter-luciferase reporter demonstrated that membrane-targeted PC1 constructs containing the membrane proximal region of the C-terminal tail, which includes the heterotrimeric G protein binding and activation domain, can stimulate NFAT luciferase activity. Inhibition of glycogen synthase kinase-3beta by LiCl treatment further increased PC1-mediated NFAT activity. PC1-mediated activation of NFAT was completely inhibited by the calcineurin inhibitor, cyclosporin A. Cotransfection of a construct expressing the Galpha(q) subunit augmented PC1-mediated NFAT activity, whereas the inhibitors of PLC (U73122) and the inositol trisphosphate and ryanodine receptors (xestospongin and 2-aminophenylborate) and a nonspecific Ca(2+) channel blocker (gadolinium) diminished PC1-mediated NFAT activity. PC2 was not able to activate NFAT. An NFAT-green fluorescent protein nuclear localization assay demonstrated that PC1 constructs containing the C-tail only or the entire 11-transmembrane spanning region plus C-tail induced NFAT-green fluorescent protein nuclear translocation. NFAT expression was demonstrated in the M-1 mouse cortical collecting duct cell line and in embryonic and adult mouse kidneys by reverse transcriptase-PCR and immunolocalization. These data suggest a model in which PC1 signaling leads to a sustained elevation of intracellular Ca(2+) mediated by PC1 activation of Galpha(q) followed by PLC activation, release of Ca(2+) from intracellular stores, and activation of store-operated Ca(2+) entry, thus activating calcineurin and NFAT.

Active Transport, Cell Nucleus↗

PubMed2004

Nicotinic acid adenine dinucleotide phosphate potentiates neurite outgrowth.

Ca(2+) regulates a spectrum of cellular processes including many aspects of neuronal function. Ca(2+)-sensitive events such as neurite extension and axonal guidance are driven by Ca(2+) signals that are precisely organized in both time and space. These complex cues result from both Ca(2+) influx across the plasma membrane and the mobilization of intracellular Ca(2+) stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca(2+) levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca(2+) from bafilomycin-sensitive Ca(2+) stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca(2+) signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca(2+) channels may therefore play important signaling roles in the nervous system.

Animals↗

PubMed2004

SHP-2 modulates interleukin-1-induced Ca2+ flux and ERK activation via phosphorylation of phospholipase Cgamma1.

Interleukin-1 (IL-1) signaling is dependent on focal adhesions, structures that are enriched with tyrosine kinases and phosphatases. Because the non-receptor tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) is enriched in focal adhesions and IL-1-induced ERK activation requires increased Ca(2+), we determined whether SHP-2 modulates IL-1-induced Ca(2+) signaling. In SHP-2-deficient fibroblasts, IL-1-induced Ca(2+) signaling and ERK activation were markedly diminished compared with cells expressing SHP-2. IL-1-induced Ca(2+) release from the endoplasmic reticulum occurred in the vicinity of focal adhesions and was strongly inhibited by the blockage of phospholipase C (PLC) catalytic activity. Immunoprecipitation and immunostaining showed that SHP-2, the endoplasmic reticulum-specific protein calnexin, and PLCgamma1 were associated with focal adhesions; however, these associations and IL-1-induced ERK activation dissipated after cells were plated on non-integrin substrates. IL-1 promoted phosphorylation of SHP-2 and PLCgamma1. IL-1-induced phosphorylation of PLCgamma1 was diminished in SHP-2-deficient cells but was restored by stable transfection with SHP-2. BAPTA/AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)) blocked IL-1-induced phosphorylation of SHP-2 and PLCgamma1, indicating mutually dependent interactive roles for Ca(2+), SHP-2, and PLCgamma1 in IL-1 signaling. We conclude that SHP-2 is critical for IL-1-induced phosphorylation of PLCgamma1 and thereby enhances IL-1-induced Ca(2+) release and ERK activation. Focal adhesions co-localizing with the endoplasmic reticulum may provide molecular staging sites required for ERK activation.

Animals↗

PubMed2005

In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061.

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

Amino Acid Substitution↗

PubMedDate not supplied

A neuromuscular screen for use in industrial toxicology.

A short objective screening technique has been developed to identify those agents that have peripheral and/or central nervous effects in small laboratory rodents (when utilized as a feature in routine testing protocols). The technique initially utilizes a series of simple quantitative and qualitative measures of various aspects of sensory and motor function. The methodology has been designed for and utilized as modular additions in a large number and variety of toxicity studies performed on industrial compounds including macrocyclic ethers, aldehydes, urethane foam catalysts, and others. It is shown to be a more sensitive early indicator of toxicity than such classical measures as body weight or clinical chemistry in those cases (such as the macrocyclic ethers and amino propionitrile) where the nervous or muscular system are the ultimate target organs. It is also a noninvasive and relatively inexpensive methodology. The second phase (a series of isolated tissue assays) establishes the ability to differentiate between reversible (pharmacologic) and irreversible (toxicologic) sensory-neural responses. The development and validation of the procedure have involved its use in both acute and subchronic studies by the inhalation, oral, ip, and sc routes. As necessary, isolated tissue and biochemical assays have been done to verify mechanisms of action. In fact, decision-tree schemes have been developed as part of the screen. These schemes allow elucidation of mechanisms of action if such is desired and statistical evaluation of the diverse data generated.

Animals↗

PubMed2006

TAR-RNA recognition by a novel cyclic aminoglycoside analogue.

The formation of the Tat-protein/TAR-RNA complex is a crucial step in the regulation of human immunodeficiency virus (HIV)-gene expression. To obtain full-length viral transcripts the Tat/TAR complex has to recruit the positive transcription elongation factor complex (P-EFTb), which interacts with TAR through its cyclin T1 (CycT1) component. Mutational studies identified the TAR hexanucleotide loop as a crucial region for contacting CycT1. Interfering with the interaction between the Tat/CycT1 complex and the TAR-RNA is an attractive strategy for the design of anti-HIV drugs. Positively charged molecules, like aminoglycosides or peptidomimetics, bind the TAR-RNA, disrupting the Tat/TAR complex. Here, we investigate the complex between the HIV-2 TAR-RNA and a neooligoaminodeoxysaccharide by NMR spectroscopy. In contrast to other aminoglycosides, this novel aminoglycoside analogue contacts simultaneously the bulge residues required for Tat binding and the A35 residue of the hexanucleotide loop. Upon complex formation, the loop region undergoes profound conformational changes. The novel binding mode, together with the easy accessibility of derivatives for the neooligoaminodeoxysaccharide, could open the way to the design of a new class of TAR-RNA binders, which simultaneously inhibit the formation of both the Tat/TAR binary complex and the Tat/TAR/CycT1 ternary complex by obstructing both the bulge and loop regions of the RNA.

Aminoglycosides↗

PubMed2004

Ryanodine receptors in human pancreatic beta cells: localization and effects on insulin secretion.

It is clear that pancreatic beta-cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca2+ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic beta cells. Using confocal microscopy, we observed RyR immunoreactivity in a vesicular pattern. RyRs did not colocalize with insulin secretory granules but partially colocalized with endosomes. Direct activation with nanomolar concentrations of ryanodine evoked increases in cytosolic Ca2+ that were coupled to transient insulin release. Insulin release stimulated by 1 nM ryanodine was sensitive to BAPTA-AM preincubation but independent of thapsigargin-sensitive endoplasmic reticulum (ER) Ca2+ pools. Blocking RyRs with micromolar concentrations of ryanodine led to BAPTA-resistant insulin release that was not associated with an increase in cytosolic Ca2+, which implicated alterations in luminal Ca2+. However, neither Ca2+ signals nor insulin release stimulated by glucose was blocked by 10-50 microM ryanodine, which suggests that the CD38/cyclic ADP-ribose/RyR pathway is not a primary mechanism of glucose action in nontransformed beta cells. We provide the first evidence that RyRs directly control insulin secretion in primary beta cells. Unexpectedly, stimulation of insulin secretion by ryanodine occurs independently of glucose and by two mechanisms, including a novel cytosolic Ca2+-independent mechanism likely involving changes in Ca2+ within the lumens of non-ER organelles, such as endosomes.

ADP-ribosyl Cyclase↗

PubMedDate not supplied

Mechanism of increased alpha-adrenoceptor-mediated contraction in small resistance arteries of rats with heart failure.

1. Alterations in a(1)-adrenoceptor signalling that result in enhanced contraction in resistance arteries in heart failure are not well characterized. To clarify whether this enhanced constriction is due to Ca(2+)-dependent or -independent effects, we measured the phenylephrine-induced changes in [Ca(2+)](i) in the presence of a Rho kinase inhibitor or an inositol 1,4,5-trisphosphate (IP(3)) receptor inhibitor. 2. Heart failure was induced in rats by ligation of the left coronary artery. Changes in the internal diameter of pressurized small femoral arteries were examined using videomicroscopy. Phenylephrine concentration-response curves, constructed in the presence of the Rho kinase inhibitor Y27632 (0.3 micromol/L) or the IP(3) receptor inhibitor xestospongin C (0.3 micromol/L), were compared in heart failure rats and sham-operated (control) rats; fura-2 Ca(2+) signals were measured in the arteries of both groups. 3. The heart : bodyweight ratio, lung : bodyweight ratio, left ventricular end-diastolic pressure and plasma B-type natriuretic peptide were significantly higher in heart failure rats compared with control rats. Phenylephrine-induced contractile responses and increases in [Ca(2+)](i) were significantly greater in arteries from heart failure rats compared with arteries from control rats. At 0.3 micromol/L, Y27632 selectively inhibited phenylephrine-induced constrictions of heart failure arteries, but had no effect on the increase in [Ca(2+)](i). 4. Immunohistochemical staining for Rho kinase was greater in heart failure rats compared with control rats. 5. The degree of inhibition of both the phenylephrine-induced constriction and the increase in [Ca(2+)](i) by xestospongin C (0.3 micromol/L) was greater in arteries from heart failure rats than in those from control rats. 6. The increased contractile response to phenylephrine in arteries of heart failure rats results from IP(3)-dependent increases in [Ca(2+)](i) and from an enhanced Ca(2+) sensitivity via a Rho kinase-dependent mechanism.

Adrenergic alpha-Agonists↗

PubMed2004

Long-term potentiation of transmitter exocytosis expressed by Ca2+-induced Ca2+ release from thapsigargin-sensitive Ca2+ stores in preganglionic nerve terminals.

We have studied whether Ca(2+)-induced Ca(2+) release (CICR) is involved in the mechanism of long-term potentiation (LTP) at nicotinic synapses of bullfrog sympathetic ganglia. Fast excitatory postsynaptic potentials (fast EPSPs) were recorded in a low-Ca(2+), high-Mg(2+) solution and quantal analysis was applied. The conditioning stimulation of the B-type preganglionic nerve at 20 Hz for 4 min consistently enhanced the amplitude and quantal content of fast EPSP for > 2 h, but only sometimes enhanced the quantal size. The LTP of quantal content produced by the conditioning tetanus was blocked by thapsigargin, a blocker of Ca(2+) pumps at Ca(2+) stores, applied before or after the conditioning tetanus, and by Xestospongin C, a blocker of inositoltrisphosphate (IP(3)) receptors, applied before the tetanus. It was not, however, blocked by ryanodine, a blocker and/or activator of ryanodine receptors, or by propranolol, a blocker of beta-adrenergic receptors. Thus the long-lasting activity of the preganglionic nerve at a high frequency causes the LTP of impulse-evoked transmitter release by the activation of CICR from thapsigargin-sensitive Ca(2+) stores in the nerve terminals. It is likely that a large Ca(2+) entry into the nerve terminals during tetanic activity primes ryanodine-insensitive Ca(2+) release channels for activation.

Adrenergic beta-Antagonists↗

PubMed2000

Protein kinase C activates non-capacitative calcium entry in human platelets.

1. In many non-excitable cells Ca2+ influx is mainly controlled by the filling state of the intracellular Ca2+ stores. It has been suggested that this store-mediated or capacitative Ca2+ entry is brought about by a physical and reversible coupling of the endoplasmic reticulum with the plasma membrane. Here we provide evidence for an additional, non-capacitative Ca2+ entry mechanism in human platelets. 2. Changes in cytosolic Ca2+ and Sr2+ were measured in human platelets loaded with the fluorescent indicator fura-2. 3. Depletion of the internal Ca2+ stores with thapsigargin plus a low concentration of ionomycin stimulated store-mediated cation entry, as demonstrated upon Ca2+ or Sr2+ addition. Subsequent treatment with thrombin stimulated further divalent cation entry in a concentration-dependent manner. 4. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol also stimulated divalent cation entry, without evoking the release of Ca2+ from intracellular stores. Cation entry evoked by thrombin or activators of PKC was abolished by the PKC inhibitor Ro-31-8220. 5. Unlike store-mediated Ca2+ entry, jasplakinolide, which reorganises actin filaments into a tight cortical layer adjacent to the plasma membrane, did not inhibit divalent cation influx evoked by thrombin when applied after Ca2+ store depletion, or by activators of PKC. Thrombin also activated Ca2+ entry in platelets in which the release from intracellular stores and store-mediated Ca2+ entry were blocked by xestospongin C. 6. These results indicate that the non-capacitative divalent cation entry pathway is regulated independently of store-mediated entry and does not require coupling of the endoplasmic reticulum and the plasma membrane. These results support the existence of a mechanism for receptor-evoked Ca2+ entry in human platelets that is independent of Ca2+ store depletion. This Ca2+ entry mechanism may be activated by occupation of G-protein-coupled receptors, which activate PKC, or by direct activation of PKC, thus generating non-capacitative Ca2+ entry alongside that evoked following the release of Ca2+ from the intracellular stores.

Blood Platelets↗

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↗

PubMed2006

Localized Ca2+ uncaging induces Ca2+ release through IP3R in smooth muscle.

AIM: Our previous study indicated that there are two types of Ca2+ release events seen in intact mouse bladder tissue. In this study our aim is to investigate the mechanism that underlies the phenomena of Ca2+ release in smooth muscle. METHODS: Single cells were isolated and tissue segments were prepared by cutting the detrusor into 0.1 cm x 0.5 cm strips running along the axis from the neck to the fundus. Single cells and intact tissue strips were co-loaded with the Ca2+ indicator and caged Ca2+ by incubation with 10 micromol/L Fluo-4 AM and DMNP-EDTA-AM. Fluo-4 AM fluorescence was detected by laser scanning confocal microscopy, and local uncaging of DMNP-EGTA was achieved by brief exposure to the output of a diode-pumped, Ti:sapphire laser tuned to 730 nm. RESULTS: Local uncaging of caged Ca2+ was able to trigger Ca2+ release events in both single cells and tissue strips from mouse bladder. The Ca2+ release events could not be blocked by ryanodine alone, but the property of the Ca2+ release was markedly altered. Surprisingly, in the presence of ryanodine, Xestospongin C completely inhibited the Ca2+ release events both in single cell and tissue experiments. CONCLUSION: (1) Two photon flash photolysis (TPFP) triggers Ca2+ induced Ca2+ release. This process involves release through type 2 ryanodine receptor channels; (2) TPFP results in the release of Ca2+ through inositol 1,4,5-trisphosphate receptors in the absence of phospholipase C activation.

Animals↗

PubMed2005

Spatial segregation of neuronal calcium signals encodes different forms of LTP in rat hippocampus.

Calcium regulates numerous processes in the brain. How one signal can coordinate so many diverse actions, even within the same neurone, is the subject of intense investigation. Here we have used two-photon calcium imaging to determine the mechanism that enables calcium to selectively and appropriately induce different forms of long-term potentiation (LTP) in rat hippocampus. Short-lasting LTP (LTP 1) required activation of ryanodine receptors (RyRs), which selectively increased calcium in synaptic spines. LTP of intermediate duration (LTP 2) was dependent on activation of inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and subsequent calcium release specifically in dendrites. Long-lasting LTP (LTP 3) was selectively dependent on L-type voltage-dependent calcium channels (L-VDCCs), which generated somatic calcium influx. Activation of NMDA receptors was necessary, but not sufficient, for the generation of appropriate calcium signals in spines and dendrites, and the induction of LTP 1 and LTP 2. These results suggest that the selective induction of different forms of LTP is achieved via spatial segregation of functionally distinct calcium signals.

Animals↗

PubMed2005

Hydroxylated xestospongins block inositol-1,4,5-trisphosphate-induced Ca2+ release and sensitize Ca2+-induced Ca2+ release mediated by ryanodine receptors.

Inositol-1,4,5-trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) often coexist within the endoplasmic/sarcoplasmic reticulum (ER/SR) membrane and coordinate precise spatial and temporal coding of Ca(2+) signals in most animal cells. Xestospongin C (XeC) was shown to selectively block IP(3)-induced Ca(2+) release and IP(3)R-mediated signaling (Gafni et al., 1997). We have further studied the specificity of xestospongin structures possessing ring hydroxyl (-OH) substituents toward IP(3)R, RyR, and ER/SR Ca(2+)-ATPase (SERCA) activities. XeC potently inhibits IP(3)R, weakly inhibits RyR1, and lacks activity toward SERCA1 and SERCA2. XeD (9-OH XeC), 7-OH-XeA, and araguspongin C isolated from the marine sponge Xestospongia species also inhibit IP(3)-mediated Ca(2+) release and lack activity toward SERCA. However, these hydroxylated derivatives possess a unique activity in that they enhance Ca(2+)-induced Ca(2+) release from SR vesicles by a mechanism involving the sensitization of RyR1 channels within the same concentration range needed to block IP(3)-induced Ca(2+) release. These results show that xestospongins and related structures lack direct SERCA inhibitory activity, as suggested by some previous studies. A new finding is that XeD and related structures possessing a hydroxylated oxaquinolizidine ring are IP(3)R blockers that also enhance Ca(2+)-induced Ca(2+) release mediated by RyRs. In intact cells, the actions of XeD are blocked by ryanodine pretreatment and do not interfere with thapsigargin-mediated Ca(2+) mobilization stemming from SERCA block. Hydroxylated bis-oxaquinolizadine derivatives isolated from Xestospongia species are novel bifunctional reagents that may be useful in ascertaining how IP(3)Rs and RyRs contribute to cell signaling.

Alkaloids↗

PubMed2001

Activation of inositol 1,4,5-trisphosphate receptor is essential for the opening of mouse TRP5 channels.

We studied the opening mechanism of Ca(2+)-permeable channels formed with mouse transient receptor potential type 5 (mTRP5) using Xenopus oocytes. After stimulation of coexpressed muscarinic M(1) receptors with acetylcholine (ACh) in a Ca(2+)-free solution, switching to 2 mM Ca(2+)-containing solution evoked a large Cl(-) current, which reflects the opening of endogenous Ca(2+)-dependent Cl(-) channels following Ca(2+) entry through the expressed channels. The ACh-evoked response was not affected by a depletion of Ca(2+) store with thapsigargin but was inhibited by preinjection of antisense oligodeoxynucleotides (ODNs) to G(q), G(11), or both. The mTRP5 channel response was also induced by a direct activation of G proteins with injection of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S). The ACh- and GTP gamma S-evoked responses were inhibited by either pretreatment with a phospholipase C inhibitor, U73122, or an inositol-1,4,5-trisphosphate (IP(3)) receptor inhibitor, xestospongin C (XeC). An activation of IP(3) receptors with injection of adenophostin A (AdA) evoked the mTRP5 channel response in a dose-dependent manner. The AdA-evoked response was not suppressed by preinjection of antisense ODNs to G(q/11) or U73122 but was suppressed by either preinjection of XeC or a peptide mimicking the IP(3) binding domain of Xenopus IP(3) receptor. These findings suggest that the activation of IP(3) receptor is essential for the opening of mTRP5 channels, and that neither G proteins, phosphoinositide metabolism, nor depletion of the Ca(2+) store directly modifies the IP(3) receptor-linked opening of mTRP5 channels.

Acetylcholine↗