scholarly journals ITPK1 mediates the lipid-independent synthesis of inositol phosphates controlled by metabolism

2019 ◽  
Vol 116 (49) ◽  
pp. 24551-24561 ◽  
Author(s):  
Yann Desfougères ◽  
Miranda S. C. Wilson ◽  
Debabrata Laha ◽  
Gregory J. Miller ◽  
Adolfo Saiardi
Keyword(s):  
Phospholipase C ◽  
Mammalian Cells ◽  
Inositol Phosphates ◽  
Phosphate Starvation ◽  
Dependent Manner ◽  
Social Amoeba ◽  
Functional Roles ◽  
Inositol Tetrakisphosphate ◽  
Independent Synthesis ◽  
Multiple Signaling

Inositol phosphates (IPs) comprise a network of phosphorylated molecules that play multiple signaling roles in eukaryotes. IPs synthesis is believed to originate with IP3 generated from PIP2 by phospholipase C (PLC). Here, we report that in mammalian cells PLC-generated IPs are rapidly recycled to inositol, and uncover the enzymology behind an alternative “soluble” route to synthesis of IPs. Inositol tetrakisphosphate 1-kinase 1 (ITPK1)—found in Asgard archaea, social amoeba, plants, and animals—phosphorylates I(3)P1 originating from glucose-6-phosphate, and I(1)P1 generated from sphingolipids, to enable synthesis of IP6. We also found using PAGE mass assay that metabolic blockage by phosphate starvation surprisingly increased IP6 levels in a ITPK1-dependent manner, establishing a route to IP6 controlled by cellular metabolic status, that is not detectable by traditional [3H]-inositol labeling. The presence of ITPK1 in archaeal clades thought to define eukaryogenesis indicates that IPs had functional roles before the appearance of the eukaryote.

scholarly journals Calcium ion dependency and the role of inositol phosphates in melatonin-induced encystment of dinoflagellates

1997 ◽  
Vol 110 (12) ◽  
pp. 1387-1393
Author(s):  
S.T. Tsim ◽  
J.T. Wong ◽  
Y.H. Wong
Keyword(s):  
Phospholipase C ◽  
Inositol Phosphates ◽  
Rank Order ◽  
Second Messengers ◽  
Signal Transduction Pathway ◽  
Dependent Manner ◽  
Functional Roles ◽  
Atpase Inhibitors ◽  
Peptide Toxin ◽  
Complex Signaling

The unicellular eukaryotic dinoflagellates shed their flagella and form a new pellicle cyst wall in response to environmental stress. This encystment process can also be induced by indoleamines such as melatonin and 5-methoxytryptamine. To decipher the complex signaling events which lead to encystment, we have investigated the functional roles of Ca2+ and inositol phosphates in indoleamine-induced encystment of the dinoflagellates Alexandrium catenella and Crypthecodinium cohnii. Pretreatment with EGTA, but not with EDTA, effectively blocked the indoleamine-induced encystment of A. catenella in a dose-dependent manner. Conversely, agents that facilitate the influx of Ca2+ (Bay K 8644, A23187 and ionomycin) dose-dependently induced encystment of A. catenella. Endoplasmic Ca2+-ATPase inhibitors such as thapsigargin and the peptide toxin melittin also induced encystment of A. catenella. These results suggest that an elevation of intracellular [Ca2+] may be involved in the encystment response. In terms of the regulation of phospholipase C, melatonin dose- and time-dependently stimulated the formation of inositol phosphates in C. cohnii. The rank order of potency for several indoleamines to stimulate inositol phosphates formation was 2-iodomelatonin > 5-methoxytryptamine > or = melatonin >> N-acetylserotonin > 5-hydroxytryptamine. This rank order was the same as for the indoleamine-induced encystment of C. cohnii as previously reported. Our results indicate that indoleamine-induced activation of phospholipase C and elevation of intracellular [Ca2+] may be proximal steps in the signal transduction pathway leading to encystment in dinoflagellates. Moreover, this is the first demonstration of the possible involvement of Ca2+ and inositol phosphates as second messengers in dinoflagellates.

Lacrimal gland inositol trisphosphate isomer and inositol tetrakisphosphate production

1990 ◽  
Vol 259 (2) ◽  
pp. G274-G281 ◽  
Author(s):  
D. A. Dartt ◽  
D. M. Dicker ◽  
L. V. Ronco ◽  
I. M. Kjeldsen ◽  
R. R. Hodges ◽  
...  
Keyword(s):  
Lacrimal Gland ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Water Soluble ◽  
Initial Increase ◽  
Dependent Manner ◽  
Cholinergic Stimulation ◽  
Maximum Increase ◽  
Concentration Dependent Manner ◽  
Inositol Tetrakisphosphate

In the lacrimal gland, cholinergic agonists stimulate protein and electrolyte/water secretion by producing inositol trisphosphate (IP3) from phosphatidylinositol bisphosphate. To determine which IP3 isomers were produced and whether inositol tetrakisphosphate (IP4) was produced during activation of secretion, rat exorbital gland acini were [3H]inositol-labeled and stimulated by the cholinergic agonist carbachol. Water-soluble inositol phosphates were separated by anion-exchange chromatography using Dowex columns or high-performance liquid chromatography. Intracellular Ca2+ concentration ([Ca2+]i) was measured by fluorescence using the Ca2+ dye fura-2. Carbachol (10(-3) M) produced a time-dependent increase in 1,4,5-IP3, 1,3,4-IP3, and 1,3,4,5-IP4 levels during 0-60 s of stimulation. The 1,4,5-IP3 level increased rapidly and was followed by a slower rise in 1,3,4-IP3 and 1,3,4,5-IP4 levels. A 3-s carbachol (10(-8) to 10(-2) M) stimulation caused a concentration-dependent rise in the 1,4,5-IP3 level. Carbachol (10(-9) to 10(-2) M) increased [Ca2+]i in a concentration-dependent manner. Carbachol (10(-3) M) increased [Ca2+]i to a maximum level by 10 s; by 60 s [Ca2+]i decreased by 38%. The maximum increase in 1,4,5-IP3 levels occurred at a higher carbachol concentration than the increase in [Ca2+]i or protein secretion. We concluded that cholinergic stimulation of the lacrimal gland rapidly increased 1,4,5-IP3 levels, which was responsible for the initial increase in [Ca2+]i and initial rapid phase of protein and fluid secretion. Cholinergic stimulation also increased 1,3,4-IP3 and 1,3,4,5-IP4, but more slowly; either acting alone or with 1,4,5-IP3, they could account for the slower phase of secretion.

scholarly journals The phospholipase C/protein kinase C pathway is involved in cathepsin G-induced human platelet activation: comparison with thrombin

1996 ◽  
Vol 313 (2) ◽  
pp. 401-408 ◽  
Author(s):  
Mustapha SI-TAHAR ◽  
Patricia RENESTO ◽  
Hervé FALET ◽  
Francine RENDU ◽  
Michel CHIGNARD
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activation ◽  
Inositol Phosphates ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Functional Responses ◽  
Kinase C

Cathepsin G, an enzyme released by stimulated polymorphonuclear neutrophils, and thrombin are two human proteinases which potently trigger platelet activation. Unlike thrombin, the mechanisms by which cathepsin G initiates platelet activation have yet to be elucidated. The involvement of the phospholipase C (PLC)/protein kinase C (PKC) pathway in cathepsin G-induced activation was investigated and compared with stimulation by thrombin. Exposure of 5-[14C]hydroxytryptamine-labelled platelets to cathepsin G, in the presence of acetylsalicylic acid and phosphocreatine/creatine kinase, induced platelet aggregation and degranulation in a concentration-dependent manner (0.1-3.0 μM). Time-course studies (0-180 s) comparing equivalent concentrations of cathepsin G (3 μM) and thrombin (0.5 unit/ml) resulted in very similar transient hydrolysis of phosphatidylinositol 4,5-bisphosphate and steady accumulation of phosphatidic acid. In addition cathepsin G, like thrombin, initiated the production of inositol phosphates. The neutrophil-derived proteinase also induced phosphorylation of both the myosin light chain and pleckstrin, a substrate for PKC, to levels similar to those observed in platelets challenged with thrombin. Inhibition of PKC by GF 109203X, a specific inhibitor, suppressed platelet aggregation and degranulation to the same extent for both proteinases. Using fura 2-loaded platelets, the rise in the cytosolic free Ca2+ concentration induced by cathepsin G was shown to result, as for thrombin, from both mobilization of internal stores and Ca2+ entry across the plasma membrane. These findings provide evidence that cathepsin G stimulates the PLC/PKC pathway as potently as does thrombin, independently of thromboxane A2 formation and ADP release, and that this pathway is required for platelet functional responses.

scholarly journals Cholera-toxin and corticotropin modulation of inositol phosphate accumulation induced by vasopressin and angiotensin II in rat glomerulosa cells

1988 ◽  
Vol 253 (3) ◽  
pp. 765-775 ◽  
Author(s):  
G Guillon ◽  
N Gallo-Payet ◽  
M N Balestre ◽  
C Lombard
Keyword(s):  
Angiotensin Ii ◽  
Cyclic Amp ◽  
Phospholipase C ◽  
Cholera Toxin ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adp Ribosylation ◽  
Glomerulosa Cells

Vasopressin (VP) and angiotensin II (AT II) stimulate the production of inositol phosphates (IP) in rat glomerulosa cells. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]), but not VP or AT II, stimulates IP production in a myo-[3H]inositol-prelabelled glomerulosa-cell membrane preparation. In combination with GTP[S], these hormones potentiate the response to GTP[S], indicating the existence of a G-protein involved in the coupling of the VP and AT II receptor with the phospholipase C. ADP-ribosylation with pertussis toxin (IAP) revealed the specific labelling of a single molecule of 41 kDa. No significant inhibition of VP- or AT II-stimulated IP accumulation was detected in intact cells when the whole 41 kDa molecule was endogenously ADP-ribosylated by IAP treatment. On the contrary, when glomerulosa cells were infected with cholera toxin (CT), both the VP- and AT II-stimulated IP accumulations were inhibited in a dose-dependent manner. Yet these effects were partial even at high concentrations of CT, and could not be related to the ADP-ribosylation of ‘alpha s’ molecules. Similarly, when the cells were infected with 1 microgram of CT/ml, the specific binding of VP and AT II decreased by 50-60%. Such results may signify that the treatment primarily affects the densities of the hormone receptors. When glomerulosa cells were incubated for 15 h in the presence of 10 nM-corticotropin (ACTH), a condition in which the intracellular concentration of cyclic AMP was increased 3-fold, the maximum IP response to 0.1 microM-VP or -AT II was decreased by 50%. When similar experiments were carried out only after a 15 min incubation period with the same concentration of ACTH, the increase in cyclic AMP was more pronounced, but no inhibition of hormone-induced IP accumulation was observed. Altogether, these results may suggest that CT exerts its action on the VP- or AT II-sensitive phospholipase C systems via a prolonged increase in intracellular cyclic AMP.

scholarly journals Inhibition of immediate-early-gene induction in renal mesangial cells by depletion of intracellular polyamines

1994 ◽  
Vol 298 (3) ◽  
pp. 647-653 ◽  
Author(s):  
E Schulze-Lohoff ◽  
H Fees ◽  
S Zanner ◽  
K Brand ◽  
R B Sterzel
Keyword(s):  
Mrna Expression ◽  
Phospholipase C ◽  
Mesangial Cells ◽  
Inositol Phosphates ◽  
Calf Serum ◽  
Inhibitory Effect ◽  
Early Gene ◽  
Immediate Early ◽  
Dependent Manner ◽  
Inhibitor Of Protein Synthesis

Mitogens have been shown to stimulate the activity of the rate-limiting enzyme for polyamine synthesis, ornithine decarboxylase (ODC), and ODC mRNA expression in cultured rat mesangial cells (MCs). In addition, inhibition of ODC by alpha-difluoromethylornithine (DFMO) results in growth arrest of MCs. To elucidate the mechanisms involved in the inhibition of MC proliferation due to polyamine depletion, we studied the effects of DFMO on the activation of phospholipase C and induction of the immediate early genes (IEGs), c-fos, c-jun and Egr-1, which are thought to regulate cell growth. Mitogenic 10% fetal-calf serum (FCS) and 1 unit/ml thrombin activated phospholipase C in MCs within 30 s, as assessed by generation of [3H]inositol phosphates. This activation was not affected by DFMO. mRNAs of the IEGs c-fos, c-jun and Egr-1 were induced by FCS within 15 min. Expression of these genes reached a peak at 60 min and disappeared at 3 h. Treatment of MCs with a growth-suppressing dose of DFMO (5 mM) inhibited mRNAs of all three IEGs by 52-87% at 1 h. Total expression of Egr-1 over 20-120 min was diminished by 41%, and the time point of maximal expression was delayed by 40 min. This inhibitory effect was abolished in a time-dependent manner (1-3 days) by prior addition of 200 microM putrescine, the reaction product of ODC. Egr-1 mRNA expression was super-induced by the inhibitor of protein synthesis, cycloheximide. This effect was also blocked by DFMO. The results indicate that the DFMO-induced process of MC growth inhibition involves steps necessary for IEG activation. The signal-transduction step sensitive to polyamines occurs distal to the activation of phospholipase C. Since reconstitution of normal induction of IEGs requires 3 days, it seems likely that polyamine depletion affects the regulation of IEG expression in an indirect fashion. We conclude that activation of IEGs requires the presence of polyamines and plays a significant role in the induction of MC replication.

scholarly journals Effect of propranolol on platelet signal transduction

1995 ◽  
Vol 309 (1) ◽  
pp. 99-104 ◽  
Author(s):  
D Dash ◽  
K Rao
Keyword(s):  
Signal Transduction ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Molecular Mechanisms ◽  
Phorbol Esters ◽  
Inositol Phosphates ◽  
Major Effect ◽  
Dependent Manner ◽  
Kinase C

Propranolol inhibits platelet secondary aggregation and secretion by mechanisms unrelated to its beta-adrenergic-blocking activity. We previously reported that a major effect of the drug is perturbation of the physical microenvironment of the human platelet membrane. To explore further the molecular mechanisms underlying propranolol-mediated platelet inhibition, we studied protein kinase C activity, estimated from the phosphorylation of the substrate protein pleckstrin, in propranolol-treated human platelets. The drug inhibited activation of the enzyme in thrombin-stimulated platelets but not in platelets stimulated with phorbol esters, indicating that its site of action might be upstream of protein kinase C. It also inhibited the activity of phospholipase C, determined from the extent of generation of inositol phosphates and phosphatidic acid, in platelets stimulated with thrombin as well as the non-hydrolysable GTP analogue guanosine 5′-[beta, gamma-imido]triphosphate in a dose-dependent manner. These data suggest that propranolol inhibits signal transduction in thrombin-stimulated platelets by interacting at the level of phospholipase C and exclude interaction of the drug with the downstream effector enzyme protein kinase C.

scholarly journals Metabolic evidence for PtdIns(4,5)P2-directed phospholipase C in permeabilized plant protoplasts

1997 ◽  
Vol 324 (1) ◽  
pp. 123-131 ◽  
Author(s):  
Charles A. BREARLEY ◽  
Paroo N. PARMAR ◽  
David E. HANKE
Keyword(s):  
Phospholipase C ◽  
Mammalian Cells ◽  
Higher Plants ◽  
Inositol Phosphates ◽  
Physiological Role ◽  
Phosphate Esters ◽  
Plant Protoplasts ◽  
Genes Encoding ◽  
Alternative Routes

Comparison of the sequences of the genes encoding phospholipase C (PLC) which have been cloned to date in plants with their mammalian counterparts suggests that plant PLC is similar to PLCδ of mammalian cells. The physiological role and mechanism of activation of PLCδ is unclear. It has recently been shown that Ins(1,4,5)P3 may not solely be the product of PtdIns(4,5)P2-directed PLC activity. Enzyme activities capable of producing Ins(1,4,5)P3 from endogenous inositol phosphates are present in Dictyostelium and also in rat liver. Significantly it has not been directly determined whether Ins(1,4,5)P3 present in higher plants is the product of a PtdIns(4,5)P2-directed PLC activity. Therefore we have developed an experimental strategy for the identification of d-Ins(1,4,5)P3 in higher plants. By the use of a short-term non-equilibrium labelling strategy in permeabilized plant protoplasts, coupled to the use of a ‘metabolic trap‘ to prevent degradation of [32P]Ins(1,4,5)P3, we were able to determine the distribution of 32P in individual phosphate esters of Ins(1,4,5)P3. The [32]Ins(1,4,5)P3 identified showed the same distribution of label in individual phosphate esters as that of [32P]PtdIns(4,5)P2 isolated from the same tissue. We thus provide in vivo evidence for the action of a PtdIns(4,5)P2-directed PLC activity in plant cells which is responsible for the production of Ins(1,4,5)P3 observed here. This observation does not, however, exclude the possibility that in other cells or under different conditions Ins(1,4,5)P3 can be generated by alternative routes.

scholarly journals Incerto-thalamic modulation of fear via GABA and dopamine

2021 ◽  
Author(s):  
Archana Venkataraman ◽  
Sarah C. Hunter ◽  
Maria Dhinojwala ◽  
Diana Ghebrezadik ◽  
JiDong Guo ◽  
...  
Keyword(s):  
Brain Regions ◽  
Zona Incerta ◽  
Dependent Manner ◽  
Post Traumatic Stress ◽  
Functional Roles ◽  
Functional Connections ◽  
Optogenetic Stimulation ◽  
Fear Generalization ◽  
Stimulation Of

AbstractFear generalization and deficits in extinction learning are debilitating dimensions of Post-Traumatic Stress Disorder (PTSD). Most understanding of the neurobiology underlying these dimensions comes from studies of cortical and limbic brain regions. While thalamic and subthalamic regions have been implicated in modulating fear, the potential for incerto-thalamic pathways to suppress fear generalization and rescue deficits in extinction recall remains unexplored. We first used patch-clamp electrophysiology to examine functional connections between the subthalamic zona incerta and thalamic reuniens (RE). Optogenetic stimulation of GABAergic ZI → RE cell terminals in vitro induced inhibitory post-synaptic currents (IPSCs) in the RE. We then combined high-intensity discriminative auditory fear conditioning with cell-type-specific and projection-specific optogenetics in mice to assess functional roles of GABAergic ZI → RE cell projections in modulating fear generalization and extinction recall. In addition, we used a similar approach to test the possibility of fear generalization and extinction recall being modulated by a smaller subset of GABAergic ZI → RE cells, the A13 dopaminergic cell population. Optogenetic stimulation of GABAergic ZI → RE cell terminals attenuated fear generalization and enhanced extinction recall. In contrast, optogenetic stimulation of dopaminergic ZI → RE cell terminals had no effect on fear generalization but enhanced extinction recall in a dopamine receptor D1-dependent manner. Our findings shed new light on the neuroanatomy and neurochemistry of ZI-located cells that contribute to adaptive fear by increasing the precision and extinction of learned associations. In so doing, these data reveal novel neuroanatomical substrates that could be therapeutically targeted for treatment of PTSD.

scholarly journals Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Benjamin N. Nelson ◽  
Savannah G. Beakley ◽  
Sierra Posey ◽  
Brittney Conn ◽  
Emma Maritz ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Cryptococcus Neoformans ◽  
Mammalian Cells ◽  
Cysteine Proteases ◽  
Dependent Manner ◽  
Life Threatening ◽  
Opportunistic Fungal Pathogen ◽  
Dose Dependent ◽  
Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

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