Pflügers Archiv

, Volume 422, Issue 6, pp 552–557 | Cite as

Thapsigargin discharges intracellular calcium stores and induces transmembrane currents in human endothelial cells

  • Marion Gericke
  • Guy Droogmans
  • Bernd Nilius
Heart, Circulation, Respiration and Blood; Environmental and Exercise Physiology


We have measured the effects of thapsigargin, a specific inhibitor of endoplasmic Ca2+-adenosine 5′-triphosphatase (Ca2+-ATPase), on membrane currents and on the intracellular Ca2+ concentration ([Ca2+]i) in single endothelial cells from the human umbilical cord vein. Currents were recorded by means of the patchclamp technique in the whole-cell mode and [Ca2+]i was measured using Fura II. Application of thapsigargin at concentrations between 0.2 and 2 μmol/l induced a slow increase in [Ca2+]i to a peak value of 400±110 nmol/l above a resting level of 120±35 nmol/l, and then slowly declined to a new steady-state level of 315±90 nmol/l (n=33). The thapsigargin-induced increase in [Ca2+]i depended on the extracellular Ca2+ concentration ([Ca2+]o: it declined after removal of extracellular Ca2+, but increased again when [Ca2+]o was augmented, indicating that the response depends on a transmembrane influx of Ca2+ ions. The peak amplitude of the histamine-induced Ca2+ transient was reduced in the presence of thapsigargin. This reduction was more pronounced when histamine was applied at the peak of the increase in [Ca2+]i induced by thapsigargin than during the rising phase of the changes in [Ca2+]i. The decline of the Ca2+ transient induced by histamine after washing out the agonist was also affected by thapsigargin. Before application of thapsigargin, this decline could be described by a single exponential with a time constant τ equal to 24.5±5 s (n=7). In the presence of thapsigargin, the decline was much slower (n =8 cells), although in four cells a fraction of about 23% still exchanged with a similar fast τ value of 29.4±4 s. Thapsigargin also induced a slowly developing inward current in endothelial cells at a holding potential of −40 mV. Voltage ramps applied before and during the development of this current indicated that a non-selective cation channel with a reversal potential near 0 mV was activated. In contrast with the Ca2+ transients, these currents did not show a declining phase. These results indicate that inhibition of the endoplasmic Ca2+ pump in endothelial cells increases [Ca2+]i. The tonic component of this increase might be partly due to opening of non-selective Ca2+-permeable cation channels activated by depletion of intracellular stores.

Key words

Endothelial cells Intracellular calcium Ca2+ stores Thapsigargin Non-selective cation channels 


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  1. 1.
    Dolor RJ, Hurwitz LM, Mirza Z, Strauss HC, Whorton AR (1992) Regulation of extracellular calcium entry in endothelial cells: role of intracellular calcium pool. Am J Physiol 262:C171-C181Google Scholar
  2. 2.
    Ely JA, Ambroz C, Baukal AJ, Christensen SB, Balla T, Catt KJ (1991) Relationship between agonistand thapsigargin-sensitive calcium pools in adrenal glomerulosa cells. J Biol Chem 266:18635–18641Google Scholar
  3. 3.
    Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450Google Scholar
  4. 4.
    Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp technique for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391:85–100Google Scholar
  5. 5.
    Horn R, Marty A (1988) Muscarinic activation of ionic currents measured by a new whole cell recording method. J Gen Physiol 92:145–159Google Scholar
  6. 6.
    Hoth M, Penner R (1992) Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355:353–356Google Scholar
  7. 7.
    Jacob (1990) Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells. J Physiol (Lond) 421:55–77Google Scholar
  8. 8.
    Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. J Clin Invest 52:2745–2756Google Scholar
  9. 9.
    Lückhoff A, Clapham DE (1992) Inositol 1,3,4,5-tetrakisphosphate activates an endothelial Ca2+-permeable channel. Nature 355:356–358Google Scholar
  10. 10.
    Mendelowitz D, Bacal K, Kunze DL (1992) Bradykinin-activated calcium influx pathway in bovine aortic endothelial cells. Am J Physiol 262:H942-H948Google Scholar
  11. 11.
    Neher E (1989) Combined Fura-2 and patch clamp measurements in rat peritoneal mast cells. In: Sellin LC, Libelius R, Thesleff S (eds) Neuromuscular junction. Elsevier, Amsterdam, pp 65–76Google Scholar
  12. 12.
    Newby A, Henderson AH (1990) Stimulus-secretion coupling in vascular endothelial cells. Annu Rev Physiol 52:661–674Google Scholar
  13. 13.
    Nilius B (1990) Permeation properties of a non-selective cation channel in human vascular endothelial cells. Pflügers Arch 416:609–611Google Scholar
  14. 14.
    Papp B, Enyedi A, Kovacs T, Sarkadi B, Wuytack F, Thastrup O, Gardos G, Bredoux R, Levy-Toledano S, Enouf J (1991) Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles. J Biol Chem 266:14593–14596Google Scholar
  15. 15.
    Putney JW Jr (1990) Capacitative calcium entry revisited. Cell Calcium 11:611–624Google Scholar
  16. 16.
    Takemura H, Hughes AR, Thastrup O, Putney JW Jr (1989) Activation of calcium entry by the tumor promoter Thapsigargin in parotid acinar cells. J Biol Chem 264:12266–12271Google Scholar
  17. 17.
    Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP (1991) Thapsigargin, a tumor promotor, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA 87:2466–2470Google Scholar
  18. 18.
    Vostalt JG, Jackson WL, Shulman NR (1991) Cytosolic and stored calcium antagonistically control tyrosine phosphorylation of specific platelet proteins. J Biol Chem 266:16911–16916Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Marion Gericke
    • 1
  • Guy Droogmans
    • 2
  • Bernd Nilius
    • 1
  1. 1.Max Planck GroupMolecular and Cellular PhysiologyLeuvenBelgium
  2. 2.Department of PhysiologyCatholic University LeuvenLeuvenBelgium

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