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Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays
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Immunoglobulin E receptor-activated calcium conductance in rat mast cells

L Zhang1, M A McCloskey

  • 1Department of Zoology and Genetics, Iowa State University, Ames 50011-3223, USA.

The Journal of Physiology
|February 15, 1995
PubMed

Insights

Antigen stimulation of immunoglobulin E (IgE) on rat basophilic leukemia (RBL-2H3) cells activates a calcium current (ICa) via capacitative calcium entry channels. This calcium influx is crucial for cell activation and degranulation.

Area of Science:

  • Immunology
  • Cell Biology
  • Biophysics

Background:

  • Immunoglobulin E (IgE) plays a critical role in allergic responses.
  • Activation of mast cells and basophils by IgE-antigen complexes triggers degranulation and mediator release.
  • Calcium influx is a key event in the signaling pathway following IgE receptor cross-linking.

Purpose of the Study:

  • To investigate the nature of the calcium current (ICa) induced by antigen stimulation in IgE-sensitized rat basophilic leukemia (RBL-2H3) cells.
  • To determine the ion selectivity and channel characteristics of the antigen-induced calcium current.
  • To elucidate the relationship between IgE receptor cross-linking, calcium influx, and capacitative calcium entry.

Main Methods:

  • Nystatin perforated-patch clamp technique to record macroscopic currents.
  • Stimulation of IgE-sensitized RBL-2H3 cells with multivalent antigen (TNP-BSA).
  • Use of various ions (Ca2+, Ba2+, Sr2+, Na+) and channel blockers (La3+, Zn2+, nitrendipine) to characterize the current.
  • Induction of calcium current using the intracellular calcium mobilizer thapsigargin.

Main Results:

  • Antigen stimulation of IgE-sensitized RBL-2H3 cells induced an inwardly rectifying calcium current (ICa) at 37°C, but not at room temperature.
  • The current was dependent on specific antigen-IgE interaction and could be carried by Ba2+ or Sr2+ in the absence of Ca2+.
  • ICa was inhibited by inorganic blockers La3+ and Zn2+, but not by cromolyn sodium or nitrendipine.
  • Thapsigargin also induced ICa, and its effect was not additive with antigen, suggesting activation of capacitative calcium entry channels.

Conclusions:

  • IgE receptor cross-linkage by antigen activates capacitative calcium entry channels in RBL-2H3 cells.
  • The antigen-induced calcium current is a non-selective cation current, primarily permeable to Ca2+, Ba2+, and Sr2+.
  • These findings provide insights into the early signaling events in IgE-mediated allergic responses.

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