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. 1999 Jul 15;341(Pt 2):401–408.

Evidence for the involvement of a small subregion of the endoplasmic reticulum in the inositol trisphosphate receptor-induced activation of Ca2+ inflow in rat hepatocytes.

R B Gregory 1, R A Wilcox 1, L A Berven 1, N C van Straten 1, G A van der Marel 1, J H van Boom 1, G J Barritt 1
PMCID: PMC1220373  PMID: 10393099

Abstract

The roles of a subregion of the endoplasmic reticulum (ER) and the cortical actin cytoskeleton in the mechanisms by which Ins(1,4,5)P3 induces the activation of store-operated Ca2+ channels (SOCs) in isolated rat hepatocytes were investigated. Adenophostin A, a potent agonist at Ins(1,4,5)P3 receptors, induced ER Ca2+ release and the activation of Ca2+ inflow. The concentration of adenophostin A that gave half-maximal stimulation of Ca2+ inflow (10 nM) was substantially lower than that (20 nM) which gave half-maximal ER Ca2+ release. A low concentration of adenophostin A (approx. 13 nM) caused near-maximal stimulation of Ca2+ inflow but only 20% of maximal ER Ca2+ release. Similar results were obtained using another Ins(1,4,5)P3-receptor agonist, 2-hydroxyethyl-alpha-d-glucopyranoside 2,3',4'-trisphosphate. Anti-type-1 Ins(1,4,5)P3-receptor monoclonal antibody 18A10 inhibited vasopressin-stimulated Ca2+ inflow but had no observable effect on vasopressin-induced ER Ca2+ release. Treatment with cytochalasin B at a concentration that partially disrupted the cortical actin cytoskeleton inhibited Ca2+ inflow and ER Ca2+ release induced by vasopressin by 73 and 45%, respectively. However, it did not substantially affect Ca2+ inflow and ER Ca2+ release induced by thapsigargin or 13 nM adenophostin A, intracellular Ca2+ release induced by ionomycin or Ins(1,4, 5)P3P4(5)-1-(2-nitrophenyl)ethyl ester ['caged' Ins(1,4,5)P3] or basal Ca2+ inflow. 1-(5-Chloronaphthalene-1-sulphonyl)homopiperazine, HCl (ML-9), an inhibitor of myosin light-chain kinase, also inhibited vasopressin-induced Ca2+ inflow and ER Ca2+ release by 53 and 44%, respectively, but had little effect on thapsigargin-induced Ca2+ inflow and ER Ca2+ release. Neither cytochalasin B nor ML-9 inhibited vasopressin-induced Ins(1,4,5)P3 formation. It is concluded that the activation of SOCs in rat hepatocytes induced by Ins(1,4,5)P3 requires the participation of a small region of the ER, which is distinguished from other regions of the ER by a different apparent affinity for Ins(1,4,5)P3 analogues and is associated with the plasma membrane through the actin skeleton. This conclusion is discussed briefly in relation to current hypotheses for the activation of SOCs.

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Selected References

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  1. Baffy G., Yang L., Raj S., Manning D. R., Williamson J. R. G protein coupling to the thrombin receptor in Chinese hamster lung fibroblasts. J Biol Chem. 1994 Mar 18;269(11):8483–8487. [PubMed] [Google Scholar]
  2. Bannikov G. A., Guelstein V. I., Montesano R., Tint I. S., Tomatis L., Troyanovsky S. M., Vasiliev J. M. Cell shape and organization of cytoskeleton and surface fibronectin in non-tumorigenic and tumorigenic rat liver cultures. J Cell Sci. 1982 Apr;54:47–67. doi: 10.1242/jcs.54.1.47. [DOI] [PubMed] [Google Scholar]
  3. Barritt G. J. Does a decrease in subplasmalemmal Ca2+ explain how store-operated Ca2+ channels are opened? Cell Calcium. 1998 Jan;23(1):65–75. doi: 10.1016/s0143-4160(98)90075-6. [DOI] [PubMed] [Google Scholar]
  4. Barritt G. J. Receptor-activated Ca2+ inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2+ signalling requirements. Biochem J. 1999 Jan 15;337(Pt 2):153–169. [PMC free article] [PubMed] [Google Scholar]
  5. Berridge M. J. Capacitative calcium entry. Biochem J. 1995 Nov 15;312(Pt 1):1–11. doi: 10.1042/bj3120001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berven L. A., Barritt G. J. A role for a pertussis toxin-sensitive trimeric G-protein in store-operated Ca2+ inflow in hepatocytes. FEBS Lett. 1994 Jun 13;346(2-3):235–240. doi: 10.1016/0014-5793(94)00481-1. [DOI] [PubMed] [Google Scholar]
  7. Berven L. A., Crouch M. F., Katsis F., Kemp B. E., Harland L. M., Barritt G. J. Evidence that the pertussis toxin-sensitive trimeric GTP-binding protein Gi2 is required for agonist- and store-activated Ca2+ inflow in hepatocytes. J Biol Chem. 1995 Oct 27;270(43):25893–25897. doi: 10.1074/jbc.270.43.25893. [DOI] [PubMed] [Google Scholar]
  8. Berven L. A., Hughes B. P., Barritt G. J. A slowly ADP-ribosylated pertussis-toxin-sensitive GTP-binding regulatory protein is required for vasopressin-stimulated Ca2+ inflow in hepatocytes. Biochem J. 1994 Apr 15;299(Pt 2):399–407. doi: 10.1042/bj2990399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bonfoco E., Leist M., Zhivotovsky B., Orrenius S., Lipton S. A., Nicotera P. Cytoskeletal breakdown and apoptosis elicited by NO donors in cerebellar granule cells require NMDA receptor activation. J Neurochem. 1996 Dec;67(6):2484–2493. doi: 10.1046/j.1471-4159.1996.67062484.x. [DOI] [PubMed] [Google Scholar]
  10. Cooper J. A. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987 Oct;105(4):1473–1478. doi: 10.1083/jcb.105.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. DeLisle S., Marksberry E. W., Bonnett C., Jenkins D. J., Potter B. V., Takahashi M., Tanzawa K. Adenophostin A can stimulate Ca2+ influx without depleting the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in the Xenopus oocyte. J Biol Chem. 1997 Apr 11;272(15):9956–9961. doi: 10.1074/jbc.272.15.9956. [DOI] [PubMed] [Google Scholar]
  12. Fagan K. A., Mons N., Cooper D. M. Dependence of the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells on capacitative Ca2+ entry. J Biol Chem. 1998 Apr 10;273(15):9297–9305. doi: 10.1074/jbc.273.15.9297. [DOI] [PubMed] [Google Scholar]
  13. Fernando K. C., Gregory R. B., Katsis F., Kemp B. E., Barritt G. J. Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes. Biochem J. 1997 Dec 1;328(Pt 2):463–471. doi: 10.1042/bj3280463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graier W. F., Paltauf-Doburzynska J., Hill B. J., Fleischhacker E., Hoebel B. G., Kostner G. M., Sturek M. Submaximal stimulation of porcine endothelial cells causes focal Ca2+ elevation beneath the cell membrane. J Physiol. 1998 Jan 1;506(Pt 1):109–125. doi: 10.1111/j.1469-7793.1998.109bx.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  16. Hajnóczky G., Lin C., Thomas A. P. Luminal communication between intracellular calcium stores modulated by GTP and the cytoskeleton. J Biol Chem. 1994 Apr 8;269(14):10280–10287. [PubMed] [Google Scholar]
  17. Hartzell H. C., Machaca K., Hirayama Y. Effects of adenophostin-A and inositol-1,4,5-trisphosphate on Cl- currents in Xenopus laevis oocytes. Mol Pharmacol. 1997 Apr;51(4):683–692. doi: 10.1124/mol.51.4.683. [DOI] [PubMed] [Google Scholar]
  18. Hofer A. M., Fasolato C., Pozzan T. Capacitative Ca2+ entry is closely linked to the filling state of internal Ca2+ stores: a study using simultaneous measurements of ICRAC and intraluminal [Ca2+]. J Cell Biol. 1998 Jan 26;140(2):325–334. doi: 10.1083/jcb.140.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holda J. R., Blatter L. A. Capacitative calcium entry is inhibited in vascular endothelial cells by disruption of cytoskeletal microfilaments. FEBS Lett. 1997 Feb 17;403(2):191–196. doi: 10.1016/s0014-5793(97)00051-3. [DOI] [PubMed] [Google Scholar]
  20. Hooser S. B., Beasley V. R., Waite L. L., Kuhlenschmidt M. S., Carmichael W. W., Haschek W. M. Actin filament alterations in rat hepatocytes induced in vivo and in vitro by microcystin-LR, a hepatotoxin from the blue-green alga, Microcystis aeruginosa. Vet Pathol. 1991 Jul;28(4):259–266. doi: 10.1177/030098589102800401. [DOI] [PubMed] [Google Scholar]
  21. Huang Y., Putney J. W., Jr Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1). J Biol Chem. 1998 Jul 31;273(31):19554–19559. doi: 10.1074/jbc.273.31.19554. [DOI] [PubMed] [Google Scholar]
  22. Liu K. Q., Bunnell S. C., Gurniak C. B., Berg L. J. T cell receptor-initiated calcium release is uncoupled from capacitative calcium entry in Itk-deficient T cells. J Exp Med. 1998 May 18;187(10):1721–1727. doi: 10.1084/jem.187.10.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lièvremont J. P., Hill A. M., Hilly M., Mauger J. P. The inositol 1,4,5-trisphosphate receptor is localized on specialized sub-regions of the endoplasmic reticulum in rat liver. Biochem J. 1994 Jun 1;300(Pt 2):419–427. doi: 10.1042/bj3000419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Miyazaki S., Yuzaki M., Nakada K., Shirakawa H., Nakanishi S., Nakade S., Mikoshiba K. Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs. Science. 1992 Jul 10;257(5067):251–255. doi: 10.1126/science.1321497. [DOI] [PubMed] [Google Scholar]
  25. Nakade S., Maeda N., Mikoshiba K. Involvement of the C-terminus of the inositol 1,4,5-trisphosphate receptor in Ca2+ release analysed using region-specific monoclonal antibodies. Biochem J. 1991 Jul 1;277(Pt 1):125–131. doi: 10.1042/bj2770125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nathanson M. H., Burgstahler A. D., Fallon M. B. Multistep mechanism of polarized Ca2+ wave patterns in hepatocytes. Am J Physiol. 1994 Sep;267(3 Pt 1):G338–G349. doi: 10.1152/ajpgi.1994.267.3.G338. [DOI] [PubMed] [Google Scholar]
  27. Palmer S., Hughes K. T., Lee D. Y., Wakelam M. J. Development of a novel, Ins(1,4,5)P3-specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P3 in unstimulated and vasopressin-stimulated rat hepatocytes. Cell Signal. 1989;1(2):147–156. doi: 10.1016/0898-6568(89)90004-1. [DOI] [PubMed] [Google Scholar]
  28. Parekh A. B., Fleig A., Penner R. The store-operated calcium current I(CRAC): nonlinear activation by InsP3 and dissociation from calcium release. Cell. 1997 Jun 13;89(6):973–980. doi: 10.1016/s0092-8674(00)80282-2. [DOI] [PubMed] [Google Scholar]
  29. Parekh A. B., Penner R. Store depletion and calcium influx. Physiol Rev. 1997 Oct;77(4):901–930. doi: 10.1152/physrev.1997.77.4.901. [DOI] [PubMed] [Google Scholar]
  30. Putney J. W., Jr, Bird G. S. The inositol phosphate-calcium signaling system in nonexcitable cells. Endocr Rev. 1993 Oct;14(5):610–631. doi: 10.1210/edrv-14-5-610. [DOI] [PubMed] [Google Scholar]
  31. Ribeiro C. M., Reece J., Putney J. W., Jr Role of the cytoskeleton in calcium signaling in NIH 3T3 cells. An intact cytoskeleton is required for agonist-induced [Ca2+]i signaling, but not for capacitative calcium entry. J Biol Chem. 1997 Oct 17;272(42):26555–26561. doi: 10.1074/jbc.272.42.26555. [DOI] [PubMed] [Google Scholar]
  32. Rossier M. F., Bird G. S., Putney J. W., Jr Subcellular distribution of the calcium-storing inositol 1,4,5-trisphosphate-sensitive organelle in rat liver. Possible linkage to the plasma membrane through the actin microfilaments. Biochem J. 1991 Mar 15;274(Pt 3):643–650. doi: 10.1042/bj2740643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saitoh M., Ishikawa T., Matsushima S., Naka M., Hidaka H. Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase. J Biol Chem. 1987 Jun 5;262(16):7796–7801. [PubMed] [Google Scholar]
  34. Sasajima H., Wang X., van Breemen C. Fractional Ca2+ release from the endoplasmic reticulum activates Ca2+ entry in freshly isolated rabbit aortic endothelial cells. Biochem Biophys Res Commun. 1997 Dec 18;241(2):471–475. doi: 10.1006/bbrc.1997.7844. [DOI] [PubMed] [Google Scholar]
  35. Takahashi M., Tanzawa K., Takahashi S. Adenophostins, newly discovered metabolites of Penicillium brevicompactum, act as potent agonists of the inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1994 Jan 7;269(1):369–372. [PubMed] [Google Scholar]
  36. Terasaki M., Song J., Wong J. R., Weiss M. J., Chen L. B. Localization of endoplasmic reticulum in living and glutaraldehyde-fixed cells with fluorescent dyes. Cell. 1984 Aug;38(1):101–108. doi: 10.1016/0092-8674(84)90530-0. [DOI] [PubMed] [Google Scholar]
  37. Watanabe H., Takahashi R., Zhang X. X., Goto Y., Hayashi H., Ando J., Isshiki M., Seto M., Hidaka H., Niki I. An essential role of myosin light-chain kinase in the regulation of agonist- and fluid flow-stimulated Ca2+ influx in endothelial cells. FASEB J. 1998 Mar;12(3):341–348. doi: 10.1096/fasebj.12.3.341. [DOI] [PubMed] [Google Scholar]
  38. Wilcox R. A., Erneux C., Primrose W. U., Gigg R., Nahorski S. R. 2-Hydroxyethyl-alpha-D-glucopyranoside-2,3',4'-trisphosphate, a novel, metabolically resistant, adenophostin A and myo-inositol-1,4,5-trisphosphate analogue, potently interacts with the myo-inositol-1,4,5-trisphosphate receptor. Mol Pharmacol. 1995 Jun;47(6):1204–1211. [PubMed] [Google Scholar]
  39. Wojcikiewicz R. J. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J Biol Chem. 1995 May 12;270(19):11678–11683. doi: 10.1074/jbc.270.19.11678. [DOI] [PubMed] [Google Scholar]
  40. Yang L., Camoratto A. M., Baffy G., Raj S., Manning D. R., Williamson J. R. Epidermal growth factor-mediated signaling of G(i)-protein to activation of phospholipases in rat-cultured hepatocytes. J Biol Chem. 1993 Feb 15;268(5):3739–3746. [PubMed] [Google Scholar]

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