scholarly journals Regulation of the localization and activity of inositol 1,4,5-trisphosphate 3-kinase B in intact cells by proteolysis

2005 ◽  
Vol 392 (3) ◽  
pp. 435-441 ◽  
Author(s):  
Jowie C. H. Yu ◽  
Samantha M. Lloyd-Burton ◽  
Robin F. Irvine ◽  
Michael J. Schell
Keyword(s):  
Endoplasmic Reticulum ◽  
Actin Cytoskeleton ◽  
Hela Cells ◽  
Second Messenger ◽  
Full Length ◽  
Intact Cells ◽  
Superior Efficacy ◽  
Inositol 1,4,5 Trisphosphate ◽  
Primary Astrocytes ◽  
Functional Relevance

IP3K (inositol 1,4,5-trisphosphate 3-kinase) catalyses the Ca2+-regulated phosphorylation of the second messenger Ins(1,4,5)P3, thereby inactivating the signal to release Ca2+ and generating Ins(1,3,4,5)P4. Here we have investigated the localization and activity of IP3KB and its modulation by proteolysis. We found that the N- and C-termini (either side of residue 262) of IP3KB localized predominantly to the actin cytoskeleton and ER (endoplasmic reticulum) respectively, both in COS-7 cells and in primary astrocytes. The functional relevance of this was demonstrated by showing that full-length (actin-localized) IP3KB abolished the histamine-induced Ca2+ response in HeLa cells more effectively than truncated constructs localized to the ER or cytosol. The superior efficacy of full-length IP3KB was also attenuated by disruption of the actin cytoskeleton. By transfecting COS-7 cells with double-tagged IP3KB, we show that the translocation from actin to ER may be a physiologically regulated process caused by Ca2+-modulated constitutive proteolysis in intact cells.

Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate

2000 ◽  
Vol 113 (8) ◽  
pp. 1365-1372 ◽  
Author(s):  
H. Niessen ◽  
H. Harz ◽  
P. Bedner ◽  
K. Kramer ◽  
K. Willecke
Keyword(s):  
Gap Junctions ◽  
Hela Cells ◽  
Second Messenger ◽  
Pancreatic Acinar Cells ◽  
Cell Coupling ◽  
Liver Parenchymal ◽  
Fura 2 ◽  
Inositol 1,4,5 Trisphosphate ◽  
Messenger Molecules ◽  
Parenchymal Cells

Intercellular propagation of signals through connexin32-containing gap junctions is of major importance in physiological processes like nerve activity-dependent glucose mobilization in liver parenchymal cells and enzyme secretion from pancreatic acinar cells. In these cells, as in other organs, more than one type of connexin is expressed. We hypothesized that different permeabilities towards second messenger molecules could be one of the reasons for connexin diversity. In order to investigate this, we analyzed transmission of inositol 1,4,5-trisphosphate-mediated calcium waves in FURA-2-loaded monolayers of human HeLa cells expressing murine connexin26, -32 or -43. Gap junction-mediated cell coupling in different connexin-transfected HeLa cells was standardized by measuring the spreading of microinjected Mn(2+) that led to local quenching of FURA-2 fluorescence. Microinjection of inositol 1,4,5-trisphosphate into confluently growing HeLa connexin32 transfectants induced propagation of a Ca(2+) wave from the injected cell to neighboring cells that was at least three- to fourfold more efficient than in HeLa Cx26 cells and about 2.5-fold more efficient than in HeLa Cx43 transfectants. Our results support the notion that diffusion of inositol 1,4,5-trisphosphate through connexin32-containing gap junctions is essential for the optimal physiological response, for example by recruiting liver parenchymal cells that contain subthreshold levels of this short lived second messenger.

scholarly journals Actions of inositol phosphates on Ca2+ pools in guinea-pig hepatocytes

1984 ◽  
Vol 224 (3) ◽  
pp. 741-746 ◽  
Author(s):  
G M Burgess ◽  
R F Irvine ◽  
M J Berridge ◽  
J S McKinney ◽  
J W Putney
Keyword(s):  
Endoplasmic Reticulum ◽  
Guinea Pig ◽  
Inositol Phosphates ◽  
Second Messenger ◽  
Release Mechanism ◽  
Specific Receptor ◽  
Rapid Release ◽  
Inositol 1,4,5 Trisphosphate

In permeabilized hepatocytes, inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate and inositol 4,5-bisphosphate induced rapid release of Ca2+ from an ATP-dependent, non-mitochondrial vesicular pool, probably endoplasmic reticulum. The order of potency was inositol 1,4,5-trisphosphate greater than inositol 2,4,5-trisphosphate greater than inositol 4,5-bisphosphate. The Ca2+-releasing action of inositol 1,4,5-trisphosphate is not inhibited by high [Ca2+], nor is it dependent on [ATP] in the range of 50 microM-1.5 mM. These results suggest a role for inositol 1,4,5-trisphosphate as a second messenger in hormone-induced Ca2+ mobilisation, and that a specific receptor is involved in the Ca2+-release mechanism.

scholarly journals Effects of elevated expression of inositol 1,4,5-trisphosphate 3-kinase B on Ca2+ homoeostasis in HeLa cells

2000 ◽  
Vol 352 (3) ◽  
pp. 709-715 ◽  
Author(s):  
Tom H. MILLARD ◽  
Peter J. CULLEN ◽  
George BANTING
Keyword(s):  
Hela Cell ◽  
Cell Line ◽  
Significant Role ◽  
Hela Cells ◽  
Second Messenger ◽  
Inducible Promoter ◽  
Hela Cell Line ◽  
Inositol 1,4,5 Trisphosphate ◽  
Elevated Expression ◽  
Stimulation Of

Ins(1,4,5)P3 3-kinase (IP3K) phosphorylates the Ca2+-mobilizing second messenger Ins(1,4,5)P3 to yield the putative second messenger Ins(1,3,4,5)P4. A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5)P3 and increased levels of Ins(1,3,4,5)P4 after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca2+ responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca2+, compared with 3% of uninduced cells. The quantity of Ca2+ within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca2+ entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5)P3 levels, and consequently in Ca2+ responses following stimulation of cells with Ins(1,4,5)P3-elevating agonists.

scholarly journals Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells

1996 ◽  
Vol 318 (2) ◽  
pp. 383-387 ◽  
Author(s):  
Jonathan M. KENDALL ◽  
Michael N BADMINTON ◽  
Graciela B. SALA-NEWBY ◽  
Anthony K CAMPBELL ◽  
ChristopherM REMBOLD
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Intact Cells ◽  
Agonist Stimulation ◽  
Novel Method ◽  
Continuous Perfusion

We describe a novel method to monitor the endoplasmic reticulum (ER) free Ca2+ in intact cells. Continuous perfusion of HeLa cells, expressing ER-targeted apoaequorin, with coelenterazine allowed the apoprotein to act as a pseudo-luciferase capable of reporting free Ca2+ from 0.1–100 µM. In intact HeLa cells, addition of ionomycin increased apoaequorin-generated light by 91%, indicating that resting ER free Ca2+ was approx. 2 µM. Agonist stimulation decreased the ER apoaequorin signal and proportionally increased cytosolic free Ca2+ consistent with agonist-induced release of Ca2+ from the ER.

scholarly journals The digitonin-permeabilized pancreatic islet model. Effect of myo-inositol 1,4,5-trisphosphate on Ca2+ mobilization

1985 ◽  
Vol 227 (3) ◽  
pp. 965-969 ◽  
Author(s):  
B A Wolf ◽  
P G Comens ◽  
K E Ackermann ◽  
W R Sherman ◽  
M L McDaniel
Keyword(s):  
Endoplasmic Reticulum ◽  
Insulin Secretion ◽  
Insulin Release ◽  
Pancreatic Islet ◽  
Second Messenger ◽  
Inositol 1 ◽  
Rapid Release ◽  
Inositol 1,4,5 Trisphosphate

Glucose-induced insulin secretion is thought to be mediated by submicromolar increases in intracellular Ca2+, although the intracellular processes are not well understood. We have used the previously characterized digitonin-permeabilized insulin-secreting pancreatic islet model to study the role of myo-inositol 1,4,5-trisphosphate (IP3), a putative second messenger for mobilization of intracellular Ca2+. Ca2+ efflux from the endoplasmic reticulum was studied with or without vanadate present to inhibit Ca2+ reuptake. IP3 (10 microM), at a free Ca2+ level of 0.06 microM, increased Ca2+ release by 30% and, when vanadate was present, by 50%. Maximal and half-maximal Ca2+ release was observed at 10 microM- and 2.5 microM-IP3, respectively. IP3 provoked a rapid release that was followed by slow reuptake. Reuptake was diminished in the presence of vanadate. Inositol 1,4-bisphosphate, inositol 1-phosphate and other phosphoinositide metabolites did not have any significant effect. Because increases in Ca2+ levels in the submicromolar range have been previously shown to induce insulin release in digitonin-permeabilized islets, our results are consistent with the concept of IP3 serving as a second messenger for insulin secretion.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

Temporal and spatial interrelationships of confronting cisternae to nuclear envelope

1992 ◽  
Vol 50 (1) ◽  
pp. 930-931
Author(s):  
John R. Palisano
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Tumor Cells ◽  
Hela Cells ◽  
Microscopic Investigation ◽  
Electron Microscopic Investigation ◽  
Serial Sections ◽  
Electron Microscopic ◽  
Fetal Rat ◽  
Temporal And Spatial

Although confronting cistemae (CC) have been observed in a variety of tumor cells and normal fetal rat, mouse, and human epithelial tissues, little is known about their origin or role in mitotic cells. While several investigators have suggested that CC arise from nuclear envelope (NE) folding back on itself during prophase, others have suggested that CC arise when fragments of NE pair with endoplasmic reticulum. An electron microscopic investigation of 0.25 um thick serial sections was undertaken to examine the origin of CC in HeLa cells.

scholarly journals Human Homologs of the Putative G Protein-Coupled Membrane Progestin Receptors (mPRα, β, and γ) Localize to the Endoplasmic Reticulum and Are Not Activated by Progesterone

2006 ◽  
Vol 20 (12) ◽  
pp. 3146-3164 ◽  
Author(s):  
Tom Krietsch ◽  
Maria Sofia Fernandes ◽  
Jukka Kero ◽  
Ralf Lösel ◽  
Maria Heyens ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
G Protein ◽  
Cell Lines ◽  
Spotted Seatrout ◽  
Membrane Receptors ◽  
Takifugu Rubripes ◽  
Camp Production ◽  
Intact Cells ◽  
Progestin Receptors ◽  
G Protein Coupled

Abstract The steroid hormone progesterone exerts pleiotrophic functions in many cell types. Although progesterone controls transcriptional activation through binding to its nuclear receptors, it also initiates rapid nongenomic signaling events. Recently, three putative membrane progestin receptors (mPRα, β, and γ) with structural similarity to G protein-coupled receptors have been identified. These mPR isoforms are expressed in a tissue-specific manner and belong to the larger, highly conserved family of progestin and adiponectin receptors found in plants, eubacteria, and eukaryotes. The fish mPRα has been reported to mediate progesterone-dependent MAPK activation and inhibition of cAMP production through coupling to an inhibitory G protein. To functionally characterize the human homologs, we established human embryonic kidney 293 and MDA-MB-231 cell lines that stably express human mPRα, β, or γ. For comparison, we also established cell lines expressing the mPRα cloned from the spotted seatrout (Cynoscion nebulosus) and Japanese pufferfish (Takifugu rubripes). Surprisingly, we found no evidence that human or fish mPRs regulate cAMP production or MAPK (ERK1/2 or p38) activation upon progesterone stimulation. Furthermore, the mPRs did not couple to a highly promiscuous G protein subunit, Gαq5i, in transfection studies or provoke Ca2+ mobilization in response to progesterone. Finally, we demonstrate that transfected mPRs, as well as endogenous human mPRα, localize to the endoplasmic reticulum, and that their expression does not lead to increased progestin binding either in membrane preparations or in intact cells. Our results therefore do not support the concept that mPRs are plasma membrane receptors involved in transducing nongenomic progesterone actions.

Apigetrin Promotes Cell Autophagy by Activating the Endoplasmic Reticulum Stress in Human Cervical Cancer Hela Cells

2021 ◽  
Vol 20 (1) ◽  
pp. 56-63
Author(s):  
Li Jiang ◽  
Zhi-Cheng Yao ◽  
Miao-Miao Liu ◽  
Run-Hui Ma ◽  
Kiran Thakur
Keyword(s):  
Cervical Cancer ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Hela Cells ◽  
Fruits And Vegetables ◽  
Radical Scavenging ◽  
Anticancer Activities ◽  
Treatment Results ◽  
Metastasis Rate ◽  
The Relationship

Cervical cancer has always been the top malignant cancer among female cancers in the world. Due to its recurrence, metastasis rate, and drug resistance, the treatment results of cervical cancer have been unsatisfactory. Apigetrin is present in a variety of fruits and vegetables and has been reported to have antioxidant, free radical scavenging, anti-inflammatory, and anticancer activities. Therefore, this study focuses on the effect of apigetrin on the autophagy of cervical cancer HeLa cells based on the previous research. The results showed that apigetrin can enhance the autophagy fluorescence of light chain 3B (LC3B), and further combined with quantitative real-time PCR (qPCR) and Western blotting found that the expression of autophagy-related genes and proteins p-mTOR, Beclin1, and LC3B increased, while the expression of AMPK, ULK1, and p62 decreased. In addition, apigetrin also promoted the release of Ca2+, the PERK/eIF2α/ATF4/chop, and IRE1α pathways activate endoplasmic reticulum (ER) stress. The addition of 4PBA proved that ER stress promoted autophagy in HeLa cells. Finally, the addition of the 3-MA indicates the relationship between autophagy and apoptosis in HeLa cells. Our results indicate that apigetrin has a certain anticancer potential and can be used as a drug adjuvant and food additive for the prevention and treatment of cervical cancer.

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