Changes In Inositol Research Articles

New alternative ingredients and genetic selection are the next game changers in rainbow trout nutrition: a metabolomics appraisal

The formulation of sustainable fish feeds based on plant ingredients supplemented by alternative ingredients to plant (insect, micro-algae, yeast) and genetic selection of fish for plant-based diets were tested on rainbow trout in two separate experiments. Plant-based diets and corresponding diets supplemented with an ingredient mix: insect, micro-algae and yeast in Experiment A, and insect and yeast in Experiment B were compared to commercial-like diets. In experiment A, the mix-supplemented diet was successful in compensating the altered growth performance of fish fed their respective plant-based diet compared to those fed the commercial diet, by restoring feed conversion. In experiment B, the selected line demonstrated improved growth performances of fish fed mix-supplemented and plant-based diets compared to the non-selected line. Metabolomics demonstrated a plasma compositional stability in fish fed mix-supplemented and basal plant-based diets comprising an amino acid accumulation and a glucose depletion, compared to those fed commercial diets. The selected line fed mix-supplemented and commercial diets showed changes in inositol, ethanol and methanol compared to the non-selected line, suggesting an involvement of microbiota. Changes in plasma glycine-betaine content in fish fed the mix-supplemented diet suggest the ability of the selected line to adapt to alternative ingredients.

Changes in three-dimensional arterial spin labeling perfusion imaging of the hippocampus in depressive Itpr2-/- mice

To study the behavioral changes of inositol 1, 4, 5-trisphosphate receptor type 2 knockout (Itpr2-/- mice) and investigate the blood perfusion changes in the hippocampus using three-dimensional arterial spin labeling (3D-ASL). 28 Itpr2-/- mice and 20 wild-type mice were assessed for depressive phenotype using behavioral tests (including sucrose consumption test, tail suspension test, forced swimming test and open field test). 15 Itpr2-/- mice and 14 wild-type mice were randomly selected for 3D-T2WI imaging of the whole brain and 3D-ASL imaging of the middle hippocampal layer, and cerebral blood flow (CBF) of the middle hippocampal layer was calculated. ITK-SNAP was used to delineate the bilateral hippocampal area and measure the average CBF value. Compared with the wild-type mice, Itpr2-/- mice exhibited a distinct depressive phenotype with significantly decreased sucrose preference (P < 0.05) and increased immobile time in tail suspension test (P < 0.05) and forced swimming test (P < 0.01), without obvious changes in the performance in open field test (P > 0.05). Significantly decreased mean CBF values were found in the left and right hippocampus of Itpr2-/- mice as compared with the wild-type mice (left: 73.30 ±5.609 vs 95.77±5.095; right: 73.53±5.700 vs 100.5±4.696; bilateral means: 73.42±5.607 vs98.12±4.754; P < 0.01). Itpr2 deficiency can cause depressive phenotype and affect the cerebral blood flow in the hippocampus of mice.

Treatment of dysphasia with rTMS and language therapy after childhood stroke: Multimodal imaging of plastic change

Expressive dysphasia accompanies left inferior frontal gyrus (IFG/Broca) injury. Recovery may relate to interhemispheric balance with homologous, contralesional IFG but is unexplored in children. We evaluated effects of inhibitory rTMS to contralesional IFG combined with intensive speech therapy (SLT). A 15year-old, right-handed male incurred a left middle cerebral artery stroke. After 30months, severe non-fluent dysphasia impacted quality of life. Language networks, neuronal metabolism and white matter pathways were explored using MRI. Language function was measured longitudinally. An intensive SLT program was combined with contralesional inhibitory rTMS of right pars triangularis. Procedures were well tolerated. Language function improved persisting to four months. Post-treatment fMRI demonstrated increased left perilesional IFG activations and connectivity at rest. Bilateral changes in inositol and glutamate metabolism were observed. Contralesional, inhibitory rTMS appears safe in childhood stroke-induced dysphasia. We observed clinically significant improvements after SLT coupled with rTMS. Advanced neuroimaging can evaluate intervention-induced plasticity.

No evidence for altered intracellular calcium-handling in airway smooth muscle cells from human subjects with asthma.

BackgroundAsthma is characterized by airway hyper-responsiveness and variable airflow obstruction, in part as a consequence of hyper-contractile airway smooth muscle, which persists in primary cell culture. One potential mechanism for this hyper-contractility is abnormal intracellular Ca2+ handling.MethodsWe sought to compare intracellular Ca2+ handling in airway smooth muscle cells from subjects with asthma compared to non-asthmatic controls by measuring: i) bradykinin-stimulated changes in inositol 1,4,5-trisphosphate (IP3) accumulation and intracellular Ca2+ concentration, ii) sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) expression, iii) mechanisms of cytoplasmic Ca2+ clearance assessed following instantaneous flash photolytic release of Ca2+ into the cytoplasm.ResultsWe found no differences in airway smooth muscle cell basal intracellular Ca2+ concentrations, bradykinin-stimulated IP3 accumulation or intracellular Ca2+ responses. Quantification of SERCA2 mRNA or protein expression levels revealed no differences in ASM cells obtained from subjects with asthma compared to non-asthmatic controls. We did not identify differences in intracellular calcium kinetics assessed by flash photolysis and calcium uncaging independent of agonist-activation with or without SERCA inhibition. However, we did observe some correlations in subjects with asthma between lung function and the different cellular measurements of intracellular Ca2+ handling, with poorer lung function related to increased rate of recovery following flash photolytic elevation of cytoplasmic Ca2+ concentration.ConclusionsTaken together, the experimental results reported in this study do not demonstrate major fundamental differences in Ca2+ handling between airway smooth muscle cells from non-asthmatic and asthmatic subjects. Therefore, increased contraction of airway smooth muscle cells derived from asthmatic subjects cannot be fully explained by altered Ca2+ homeostasis.

Conformational Stability of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Dictates Its Substrate Selectivity

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPK1) converts inositol 1,3,4,5,6-pentakisphosphate(IP5) to inositol hexakisphosphate (IP6). IPK1 shares structural similarity with protein kinases and is suspected to employ a similar mechanism of activation. Previous studies revealed roles for the 1- and 3-phosphates of IP5 in IPK1 activation and revealed that the N-lobe of IPK1 is unstable in the absence of inositol phosphate (IP). Here, we demonstrate the link between IPK1 substrate specificity and the stability of its N-lobe. Limited proteolysis of IPK1 revealed that N-lobe stability is dependent on the presence of the 1-phosphate of the substrate, whereas overall stability of IPK1 was increased in ternary complexes with nucleotide and IPs possessing 1- and 3-phosphates that engage the N-lobe of IPK1. Thus, the 1- and 3-phosphates possess dual roles in both IPK1 activation and IPK1 stability. To test whether kinase stability directly contributed to substrate selectivity of the kinase, we engineered IPK1 mutants with disulfide bonds that artificially stabilized the N-lobe in an IP-independent manner thereby mimicking its substrate-bound state in the absence of IP. IPK1 E82C/S142C exhibited a DTT-sensitive 5-fold increase in kcat for 3,4,5,6-inositol tetrakisphosphate (3,4,5,6-IP4) as compared with wild-type IPK1. The crystal structure of the IPK1 E82C/S142C mutant confirmed the presence of the disulfide bond and revealed a small shift in the N-lobe. Finally, we determined that IPK1 E82C/S142C is substantially more stable than wild-type IPK1 under nonreducing conditions, revealing that increased stability of IPK1 E82C/S142C correlates with changes in substrate specificity by allowing IPs lacking the stabilizing 1-phosphate to be used. Taken together, our results show that IPK1 substrate selection is linked to the ability of each potential substrate to stabilize IPK1.

Roles of Phosphate Recognition in Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase (IPK1) Substrate Binding and Activation

Inositol phosphate kinases (IPKs) sequentially phosphorylate inositol phosphates (IPs) to yield a group of small signaling molecules involved in diverse cellular processes. IPK1 (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) phosphorylates inositol 1,3,4,5,6-pentakisphosphate to inositol 1,2,3,4,5,6-hexakisphosphate; however, the mechanism of IP recognition employed by IPK1 is currently unresolved. We demonstrated previously that IPK1 possesses an unstable N-terminal lobe in the absence of IP, which led us to propose that the phosphate profile of the IP was linked to stabilization of IPK1. Here, we describe a systematic study to determine the roles of the 1-, 3-, 5-, and 6-phosphate groups of inositol 1,3,4,5,6-pentakisphosphate in IP binding and IPK1 activation. The 5- and 6-phosphate groups were the most important for IP binding to IPK1, and the 1- and 3-phosphate groups were more important for IPK1 activation than the others. Moreover, we demonstrate that there are three critical residues (Arg-130, Lys-170, and Lys-411) necessary for IPK1 activity. Arg-130 is the only substrate-binding N-terminal lobe residue that can render IPK1 inactive; its 1-phosphate is critical for full IPK1 activity and for stabilization of the active conformation of IPK1. Taken together, our results support the model for recognition of the IP substrate by IPK1 in which (i) the 4-, 5-, and 6-phosphates are initially recognized by the C-terminal lobe, and subsequently, (ii) the interaction between the 1-phosphate and Arg-130 stabilizes the N-terminal lobe and activates IPK1. This model of IP recognition, believed to be unique among IPKs, could be exploited for selective inhibition of IPK1 in future studies that investigate the role of higher IPs.

Defining protein kinase/phosphatase isoenzymic regulation of mGlu5 receptor‐stimulated phospholipase C and Ca2+ responses in astrocytes

Cyclical phosphorylation and dephosphorylation of a key residue within the C-terminal domain of the activated type 5 metabotropic glutamate (mGlu₅) receptor is believed to cause the synchronous, oscillatory changes in inositol 1,4,5-trisphosphate and Ca²⁺ levels observed in a variety of cell types. Here, we have attempted to better define the kinase and phosphatase enzymes involved in this modulation. Ca²⁺ and [³H]inositol phosphate ([³H]IP(x) ) measurements in astrocyte preparations have been used to evaluate the effects of pharmacological inhibition of protein kinase C (PKC) and protein phosphatase activities and small interfering RNA-mediated specific PKC isoenzymic knock-down on mGlu₅ receptor signalling. Ca²⁺ oscillation frequency or [³H]IP(x) accumulation in astrocytes stimulated by mGlu₅ receptors, was concentration-dependently decreased by protein phosphatase-1/2A inhibition or by PKC activation. PKC inhibition also increased [³H]IP(x) accumulation two- to threefold and changed the Ca²⁺ response into a peak-plateau response. However, selective inhibition of conventional PKC isoenzymes or preventing changes in [Ca²⁺](i) concentration by BAPTA-AM loading was without effect on mGlu₅ receptor-stimulated [³H]IP(x) accumulation. Selective knock-down of PKCδ was without effect on glutamate-stimulated Ca²⁺ responses; however, selective PKCε knock-down in astrocytes changed Ca²⁺ responses from oscillatory into peak-plateau type. These data confirm the acute regulation of mGlu₅ receptor signalling by protein kinases and protein phosphatases and provide novel data pinpointing the isoenzymic dependence of this regulation in the native mGlu₅ receptor-expressing rat cortical astrocyte. These data also highlight a potential alternative mechanism by which mGlu₅ receptor signalling might be therapeutically manipulated.

Site directed mutagenesis to the rescue: unravelling conformational changes of inositol 1,3,4,5,6-pentakisphosphate 2-kinase

Site directed mutagenesis to the rescue: unravelling conformational changes of inositol 1,3,4,5,6-pentakisphosphate 2-kinase

Expression of inositol 1,4,5-triphosphate receptor type I in the myocardial tissue of rats with endotoxic shock

Objective To investigate the changes of inositol 1,4,5-triphosphate receptor type I （ IP3 R I ） expression in the myocardial tissue of rats with endotoxic shock and probe the possible mechanisms of myocardial depression. Methods Thirty-three male Wistar rats were divided into four groups：control group （n = 6 ） were injected normal saline（ 4 ml/kg ）, and the other 3 groups （n = 9 ） were injected endotoxin via femoral vein with a different dose of 10 mg/kg, 5 mg/kg, and the artificial sacrificed group （ 10 mg/kg, the rats were sacrificed when the mean arterial pressure was first rapidly decreased, then gradually increased to the normal level）. IP3R I expression in the myocardial tissue of rats was detected by immunohistochemistry, Western blot and colloidal gold immunoelectron microscopy technique. Results IP3RIexpressions were increased significantly in each group of rats with endotoxic shock than in the control group （ P 〈 0. 01 ）, and most obviously enhanced in the the artificial sacrificed group. The expression of IP3RIwas related to the level of mean arterial pressure. Ectopic expression of IP3RIwas observed around the cell membrane of the cardiac myocytes. Conclusion （ 1 ） The expression of IP3 Rlenhances in the myocardial tissue of rats with endotoxic shock, and exists ectopic expression. （2） The expression of IP3 RIwas related to the level of mean arterial pressure. （3） IP3RI may be related to the cardiac contractility and involved in the regulation of mean arterial pressure. Key words: Endotoxic shock ; Myocardium ; Inositol 1,4,5-triphosphate receptor type I ; Rat

Blue light induced changes in inositol 1,4,5-trisphosphate inCuscuta campestrisseedlings

Previous studies revealed that blue light stimulates and red light inhibits prehaustoria development in young seedlings of the parasitic weed Cuscuta campestris (field dodder). This study showed a positive correlation between blue light induced increases in inositol 1,4,5-trisphosphate (IP3) and blue light-mediation of prehaustoria development, prior to host attachment. Blue light induced a significant increase in the level of IP3, with a peak at about 30 min. Thereafter, the level of IP3 declined to the resting value after 2 h of blue light. Irradiation with 10 min red light pulse applied directly at the end of each 0.05, 0.1, 0.15, 0.2, 0.3, 1, 2 and 4 h blue light significantly reduced IP3, while high levels of IP3 were observed after 10 min far-red pulse. The G-protein inhibitor pertussis toxin inhibited prehaustoria developed under blue light, suggesting that receptor-coupled G-proteins are likely to be involved in prehaustoria development. These results are the first in vivo demonstration of a possible role for IP3 as a second messenger in the blue light signal transduction process in prehaustoria development in Cuscuta.

Cross-linking of FcɛRI causes Ca 2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca 2+ response evoked by cross-linking of the high affinity receptors for IgE (FcɛRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcɛRI-mediated Ca 2+ response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP 3) production. This suggests that the Ca 2+ signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cγ was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca 2+ pathway to be dependent on clathrin.

Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture

Phenotypic modulation of vascular myocytes is important for vascular development and adaptation. A characteristic feature of this process is alteration in intracellular Ca(2+) handling, which is not completely understood. We studied mechanisms involved in functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine (Ry)-sensitive Ca(2+) stores, store-operated Ca(2+) entry (SOCE), and receptor-operated Ca(2+) entry (ROCE) associated with arterial myocyte modulation from a contractile to a proliferative phenotype in culture. Proliferating, cultured myocytes from rat mesenteric artery have elevated resting cytosolic Ca(2+) levels and increased IP(3)-sensitive Ca(2+) store content. ATP- and cyclopiazonic acid [CPA; a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium are significantly larger in proliferating arterial smooth muscle cells (ASMCs) than in freshly dissociated myocytes, whereas caffeine (Caf)-induced Ca(2+) release is much smaller. Moreover, the Caf/Ry-sensitive store gradually loses sensitivity to Caf activation during cell culture. These changes can be explained by increased expression of all three IP(3) receptors and a switch from Ry receptor type II to type III expression during proliferation. SOCE, activated by depletion of the IP(3)/CPA-sensitive store, is greatly increased in proliferating ASMCs. Augmented SOCE and ROCE (activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol) in proliferating myocytes can be attributed to upregulated expression of, respectively, transient receptor potential proteins TRPC1/4/5 and TRPC3/6. Moreover, stromal interacting molecule 1 (STIM1) and Orai proteins are upregulated in proliferating cells. Increased expression of IP(3) receptors, SERCA2b, TRPCs, Orai(s), and STIM1 in proliferating ASMCs suggests that these proteins play a critical role in an altered Ca(2+) handling that occurs during vascular growth and remodeling.

ABNORMALITIES OF SARCOPLASMIC RETICULUM Ca2+ MOBILIZATION IN AORTIC SMOOTH MUSCLE CELLS FROM STREPTOZOTOCIN‐INDUCED DIABETIC RATS

1. Previously, we found that contractions in response to receptor-dependent (i.e. a(1)-adrenoceptor agonist phenylephrine) and -independent (i.e. cyclopiazonic acid) stimuli are decreased in rat aorta during late diabetes. The aim of the present study was to further investigate the changes of intracellular Ca(2+) homeostasis in diabetic aortic smooth muscle cells. Functional changes of inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores of the sarcoplasmic reticulum (SR) were evaluated using Fluo-3 acetoxymethyl ester fluorescence, western blot and organ bath techniques. 2. In aortic smooth muscle cells from diabetic rats, the Ca(2+) release and Ca(2+) influx caused by both 10 mmol/L phenylephrine (depletion of IP(3)-sensitive Ca(2+) stores) and 1 mmol/L ryanodine (depletion of ryanodine-sensitive Ca(2+) stores) were both significantly decreased compared with control. Moreover, protein expression levels of IP(3) (260 kDa) and ryanodine receptors (500 kDa) were reduced by 31.8 +/- 7.7 and 69.2 +/- 8.4%, respectively, in aortas from diabetic rats compared with those from control rats. 3. In diabetic rat aorta, phenylephrine-induced contractility was decreased to approximately two-thirds of that in controls, whereas ryanodine alone did not cause obvious contraction in aortas from either control or diabetic rats. 4. The present results suggest that the hyporeactivity of aortic smooth muscle to vasoconstrictors in diabetes results mainly from changes to the IP(3)-sensitive Ca(2+) release pathway. The SR Ca(2+) signalling pathway plays a crucial role in the development of diabetic vascular complications.

Changes in starch and inositol 1,4,5‐trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.

Single Cell Analysis and Temporal Profiling of Agonist-mediated Inositol 1,4,5-Trisphosphate, Ca2+, Diacylglycerol, and Protein Kinase C Signaling using Fluorescent Biosensors

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.

Specificity of G Protein-Coupled Receptor Kinase 6-Mediated Phosphorylation and Regulation of Single-Cell M3Muscarinic Acetylcholine Receptor Signaling

Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1alpha (K46RCK1alpha) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells.

Two-state Conformational Changes in Inositol 1,4,5-Trisphosphate Receptor Regulated by Calcium

Inositol 1,4,5-trisphosphate receptor (IP3R) is a highly controlled calcium (Ca2+) channel gated by inositol 1,4,5-trisphosphate (IP3). Multiple regulators modulate IP3-triggered pore opening by binding to discrete allosteric sites within IP3R. Accordingly we have postulated that these regulators structurally control ligand gating behavior; however, no structural evidence has been available. Here we show that Ca2+, the most pivotal regulator, induced marked structural changes in the tetrameric IP3R purified from mouse cerebella. Electron microscopy of the IP3R particles revealed two distinct structures with 4-fold symmetry: a windmill structure and a square structure. Ca2+ reversibly promoted a transition from the square to the windmill with relocations of four peripheral IP3-binding domains, assigned by binding to heparin-gold. Ca2+-dependent susceptibilities to limited digestion strongly support the notion that these alterations exist. Thus, Ca2+ appeared to regulate IP3 gating activity through the rearrangement of functional domains.

Microarray analysis of brain RNA in mice with methylenetetrahydrofolate reductase deficiency and hyperhomocysteinemia

Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common genetic cause of hyperhomocysteinemia, which is associated with increased risk for cardiovascular disease, stroke and possibly other neurological disorders. Microarray analysis of brain RNA from day 14 Mthfr −/− mice revealed several genes with altered expression. Expression changes in inositol 1,4,5-triphosphate receptor, type 1 ( Itpr1), proteolipid protein ( Plp), neurogenic differentiation factor 1 ( Neurod1), S100 calcium binding protein A8 ( S100a8), and methylenetetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase ( Mthfd2) were confirmed by RT-PCR. We propose that neuronal damage by hyperhomocysteinemia may involve disruption of intracellular calcium.

Hyperosmotic stress induces a rapid and transient increase in inositol 1,4,5-trisphosphate independent of abscisic acid in Arabidopsis cell culture.

Phospholipid metabolism is involved in hyperosmotic-stress responses in plants. To investigate the role of phosphoinositide-specific phospholipase C (PI-PLC)-a key enzyme in phosphoinositide turnover-in hyperosmotic-stress signaling, we analyzed changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in response to hyperosmotic shock or salinity in Arabidopsis thaliana T87 cultured cells. Within a few s, a hyperosmotic shock, caused by mannitol, NaCl, or dehydration, induced a rapid and transient increase in Ins(1,4,5)P3. However, no transient increase was detected in cells treated with ABA. Neomycin and U73122, inhibitors of PI-PLC, inhibited the increase in Ins(1,4,5)P3 caused by the hyperosmotic shock. A rapid increase in phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in response to the hyperosmotic shock also occurred, but the rate of increase was much slower than that of Ins(1,4,5)P3. These findings indicate that the transient Ins(1,4,5)P3 production was due to the activation of PI-PLC in response to hyperosmotic stress. PI-PLC inhibitors also inhibited hyperosmotic stress-responsive expression of some dehydration-inducible genes, such as rd29A (lti78/cor78) and rd17 (cor47), that are controlled by the DRE/CRT cis-acting element but did not inhibit hyperosmotic stress-responsive expression of ABA-inducible genes, such as rd20. Taken together, these results suggest the involvement of PI-PLC and Ins(1,4,5)P3 in an ABA-independent hyperosmotic-stress signal transduction pathway in higher plants.

Reduced [ 3H]IP 3 binding but unchanged IP 3 receptor levels in the rat hippocampus CA1 region following transient global ischemia and tolerance induction

Changes in inositol (1,4,5)-trisphosphate (IP 3) binding properties and the protein level of the IP 3 receptor have been reported in different pathological conditions in the brain, e.g. cerebral ischemia, Alzheimer’s disease, and Huntingtons disease. We used the 4-vessel occlusion model in rat brain to investigate the effect of transient ischemica insults on the IP 3 receptor mRNA level, the IP 3 receptor protein level and [ 3H]IP 3 binding. Recirculation periods were limited (1–72 h) to avoid the development of delayed neuronal death. We found that the IP 3 receptor mRNA levels were decreased after damage-inducing ischemia (9 min) in the hippocampus CA1 and CA3 regions. The mRNA levels were unaltered after tolerance-inducing ischemia (3 min). However, [ 3H]IP 3 binding was significantly reduced after both damage- and tolerance-inducing ischemia in the hippocampus CA1 region. Furthermore, all investigated brain areas showed a decreased [ 3H]IP 3 binding when tolerance-inducing ischemia was followed by a second ischemic insult (3+8.5 min ischemia). The IP 3 receptor protein levels remained constant in all investigated brain areas. These results indicate that a reduced [ 3H]IP 3 binding capability in the particularly vulnerable areas occurs as an early consequence of cerebral ischemia, before IP 3 receptor protein levels are reduced in these areas. Structural or conformational changes altering IP 3 binding may be of necessity on the pathway leading to down-regulation of IP 3 receptor protein levels, as observed by others.