U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation

  • J. P. Wang
Original Articles


1-[6-[[17a-3-Methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca2+ concentration ([Ca 2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca2+]i elevation of neutrophils suspended in Ca2+-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC50 values 4.06 ± 0.27 µM and 4.04 ± 0.44 µM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC50 value 0.62 ± 0.04 µM) U-73122 also reduced the intracellular Ca2+ mobilization of neutrophils suspended in Ca 2+-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC50 values 0.52 ± 0.02 µM and 6.82 ± 0.74 µM, respectively. 1-[6-[[17f3-Methoxyestra-1,3,5(10)-trien-l7-yl]amino]hexyl]2,5-pyrrolidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca2+]i elevation of neutrophils challenged by fMLP in Ca2+-containing medium, but not in Ca2+-free medium, with IC50 value 22.30 ± 1.61 µM. In Mn2+-quench studies, U-73122 suppressed the Mn2+ influx in CPA-activated neutrophils (IC50 value was 7.16 ± 0.28 µM) as well as in resting neutrophils (IC50 value was 6.72 ± 0.30 μM). U-73343 also suppressed the Mn2+ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca2+]i of activated neutrophils is attributed partly to the suppression of Ca2+ release from the intracellular Ca2+ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca2+ entry from the extracellular space through PLC-independent processes.

Key words

Neutrophil (rat) Ca2+ influx U-73122 Phospholipase C inhibition U-73343 Cytosolic Ca2+ concentration Mn2+-quench 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andersson T, Schlegel W, Monod A, Krause KH, Stendahl O, Lew DP (1986) Leukotriene B4 stimulation of phagocytes results in the formation of inositol 1,4,5-trisphosphate. A second messenger for Ca2+ mobilization. Biochem J 240:333–340Google Scholar
  2. Baggiolini M, Dewald B (1985) The neutrophil. Int Arch Allergy Appl Immunol 76 [Suppl] 1:13–20Google Scholar
  3. Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signalling. Nature 341:197–205Google Scholar
  4. Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S (1990) Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther 255:756–768Google Scholar
  5. Borregaard N (1988) The human neutrophil. Function and dysfunction. Eur J Haematol 41:401–413Google Scholar
  6. Böyum A (1968) Isolation of mononuclear cells and granulocytes from blood. Scand J Clin Invest 97 [Suppl]:77–89Google Scholar
  7. Burgess GM, McKinney JS, Irvine RF, Berridge MJ, Hoyle PC, Putney JW Jr (1984) Inositol 1,4,5-trisphosphate may be a signal for f-Met-Leu-Phe induced intracellular Ca mobilization in human leucocytes (HL-60 cells). FEBS Lett 176:193–196Google Scholar
  8. Chen TH, Lee B, Hsu WH (1994) Arginine vasopressin-stimulated insulin secretion and elevation of intracellular Ca++ concentration in rat insulinoma cells: influences of a phospholipase C inhibitor 1-[6-[[17β-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U-73122) and a phospholipase A2 inhibitor N-(p-amylcinnamoyl)anthranilic acid. J Pharmacol Exp Ther 270:900–904Google Scholar
  9. Demaurex N, Lew DP, Krause KH (1992) Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells. J Biol Chem 267:2318–2324Google Scholar
  10. Di Virgilio F, Vicentini LM, Treves S, Riz G, Pozzan T (1985) Inositol phosphate formation in fMet-Leu-Phe-stimulated human neutrophils does not require an increase in the cytosolic free Ca2+ concentration. Biochem J 229:361–367Google Scholar
  11. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of calcium indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450Google Scholar
  12. Hawes BE, Waters SB, Janovick JA, Bleasdale JE, Conn PM (1992) Gonadotropin-releasing hormone-stimulated intracelluar Ca2+ fluctuations and luteinizing hormone release can be uncoupled from inositol phosphate production. Endocrinology 130: 3475–3483Google Scholar
  13. Jin W, Lo TM, Loh HH, Thayer SA (1994) U-73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells. Brain Res 642:237–243Google Scholar
  14. Krause KH, Pittet D, Volpe P, Pozzan T, Meldolesi J, Lew DP (1989) Calciosome, a sarcoplasmic reticulum-like organelle involved in the intracellular Ca2+ handling by non-muscle cells: studies in human neutrophils and HL-60 cells. Cell Calcium 10:351–361Google Scholar
  15. Lagast H, Pozzan T, Waldvogel FA, Lew DP (1984) Phorbol myristate acetate stimulates ATP-dependent calcium transport by the plasma membrane of neutrophils. J Clin Invest 73:878–883Google Scholar
  16. Liu CM, Hermann TE (1978) Characterization of ionomycin as a calcium ionophore. J Biol Chem 253:5892–5894Google Scholar
  17. Meldolesi J, Clementi E, Fasolato C, Zacchetti D, Pozzan T (1991) Ca2+ influx following receptor activation. Trends Pharmacol Sci 12:289–292Google Scholar
  18. Meyer T, Holowka D, Stryer L (1988) Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate. Science 240:653–656Google Scholar
  19. Parekh AB, Terlau H, Stühmer W (1993) Depletion of insP3 stores activates a Ca2+ and K+ current by means of a phosphatase and a diffusible messenger. Nature 364:814–818Google Scholar
  20. Pozzan T, Lew DP, Wollheim CB, Tsien RY (1983) Is cytosolic ionized calcium regulating neutrophil activation? Science 221:1413–1415Google Scholar
  21. Putney JW Jr (1990) Capacitative calcium entry revisited. Cell Calcium 11:611–624Google Scholar
  22. Putney JW Jr, Takemura H, Hughes AR, Horstman DA, Thastrup O (1989) How do inositol phosphates regulate calcium signaling? FASEB J 3:1899–1905Google Scholar
  23. Randriamampita C, Tsien RY (1993) Emptying of intracellular Ca2+ stores releases a novel small messenger that stimulates Ca2+ influx. Nature 364:809–814Google Scholar
  24. Sage SO, Merritt JE, Hallam TJ, Rink TJ (1989) Receptor-mediated calcium entry in fura-2-loaded human platelets stimulated with ADP and thrombin. Dual wavelengths studies with Mn2+. Biochem J 258:923–926Google Scholar
  25. Seidler NW, Jona I, Vegh M, Martonosi A (1989) Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum. J Biol Chem 264:17816–17823Google Scholar
  26. Shibuya I, Douglas WW (1992) Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium, and lanthanum, but not to nickel: evidence provided by fluorescence changes in fura-2-loaded cells. Endocrinology 131:1936–1941Google Scholar
  27. Smith PM, Gallacher DV (1994) Thapsigargin-induced Ca2+ mobilization in acutely isolated mouse lacrimal acinar cells is dependent on a basal level of ins(1,4,5)P3 and is inhibited by heparin. Biochem J 299:37–40Google Scholar
  28. Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE (1990) Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther 253:688–697Google Scholar
  29. Tallarida RJ, Murray RB (1987) Manual of pharmacological calculations with computer programs. Springer, New York, Berlin Heidelberg, pp 16–18Google Scholar
  30. Thompson AK, Mostafapour SP, Denlinger LC, Bleasdale JE, Fisher SK (1991) The aminosteroid U-73122 inhibits muscarinic receptor sequestration and phosphoinositide hydrolysis in SKN-SH neuroblastoma cells. A role for Gp in receptor compartmentation. J Biol Chem 266:23856–23862Google Scholar
  31. Verghese MW, Smith CD, Snyderman R (1986) Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants. J Cell Biochem 32:59–69Google Scholar
  32. Vickers JD (1993) U-73122 affects the equilibria between the phosphoinositides as well as phospholipase C activity in unstimulated and thrombin-stimulated human and rabbit platelets. J Pharmacol Exp Ther 266:1156–1163Google Scholar
  33. von Tscharner V, Prod'hom B, Baggiolini M, Reuter H (1986) Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration. Nature 324:369–372Google Scholar
  34. Wang XD, Kiang JG, Smallridge RC (1994a) A phospholipase C inhibitor, U73122, blocks TSH-induced inositol trisphosphate production, Ca2+ increase and arachidonic acid release in FRTL-5 thyroid cells. Biochim Biophys Acta 1223:101–106Google Scholar
  35. Wang JP, Raung SL, Hsu MF, Chen CC (1994b) Inhibition by gomisin C (a lignan from Schizandra chinensis) of the respiratory burst of rat neutrophils. Br J Pharmacol 113:945–953Google Scholar
  36. Wang JP, Raung SL, Kuo YH, Teng CM (1995) Daphnoretininduced respiratory burst in rat neutrophils is, probably, mainly through protein kinase C activation. Eur J Pharmacol 288: 341–348Google Scholar
  37. Wong K, Kwan-Yeung L, Turkson J (1992) Staurosporine clamps cytosolic free Ca2+ concentrations of human neutrophils. Biochem J 283:499–505Google Scholar
  38. Yule DI, Williams JA (1992) U-73122 inhibits Ca2+ oscillations in response to cholecytokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells. J Biol Chem 267:13830–13835Google Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  • J. P. Wang
    • 1
  1. 1.Department of Medical ResearchTaichung Veterans General Hospital, 160TaichungRepublic of China

Personalised recommendations