Mast cell activation involves plasma membrane potential‐ and thapsigargin‐sensitive intracellular calcium pools

Abstract

The regulation and role of the intracellular Ca2+ pools were studied in rat peritoneal mast cells. Cytosolic free calcium concentration ([Ca2+]i) was monitored in fura-2 loaded mast cells. In the presence of Ca2+ and K+, compound 48/80 induced a biphasic increase in [Ca2+]i composed of a fast transient phase and an apparent sustained phase. The sustained phase was partially inhibited by the addition of Mn2+. DTPA, a cell-impermeant chelator of Mn2+, reversed this inhibition, suggesting that a quenching of fura-2 fluorescence occurs in the extracellular medium. In the absence of extracellular Ca2+, the transient phase, but not the sustained one, could be preserved, provided that mast cells were depolarized. The transient phase was completely abolished by thapsigargin, a microsomal Ca(2+)-ATPase inhibitor. Maximum histamine release induced by either compound 48/80 or antigen was obtained in the absence of added Ca2+ only when mast cells were depolarized. These histamine releases were inhibited by low doses (< 30 nM) of thapsigargin. Thapsigargin at higher doses induced histamine release which was unaffected by changing the plasma membrane potential, but was completely dependent on extracellular Ca2+, showing that a Ca2+ influx is required for thapsigargin-induced exocytosis. Together, these results suggest that the mobilization of Ca2+ from thapsigargin sensitive-intracellular pools induced by compound 48/80 or antigen is sufficient to trigger histamine release. The modulation of these pools by the plasma membrane potential suggest their localization is close to the plasma membrane.

Effect of NMDA receptor ligands on mast cell histamine release, a reappraisal

“…This observation is in line with previous results dealing with the secretory effect induced by the synthetic polyamines methoctramine and compound 48/80 or by cationic peptides (Church et al 1991;Kassel et al 1995;. In contrast, Purcell et al (1996) indicated that the secretory effect of spermine, but not that of compound 48/80, was dependent upon extracellular calcium, but results were not shown and the method used was not indicated.…”

Activation of CD47 receptors causes histamine secretion from mast cells

“…6A). Since lipid rafts are also involved in the exocytotic process [48], a potential inhibitory effect of MbCD on secretory granule fusion with the plasma membrane was assessed by pretreating cells with MbCD prior to stimulation with the Ca 2+ ionophore A23187 [34]. No significant inhibition of A23187-induced exocytosis was found after pretreatment with MbCD (Fig.…”

“…Intracellular calcium was measured from Fura-2-loaded mast cell suspensions using a spectrofluorimeter (Hitachi F-2000) essentially as previously described [34]. Briefly, mast cells (110 6 cells/ml) were incubated with 1 mM Fura-2/AM for 15 min at room temperature in HEPES buffer.…”

Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt‐dependent pathway

“…Intracellular calcium was measured from Fura-2-loaded astrocytoma cell suspensions using a spectrofluorimeter (Hitachi F-2000) essentially as previously described (Kassel et al, 1995;Lynch et al, 1994). Briefly, astrocytoma cells (1 3 10 6 cells/mL) were incubated with 1 lM Fura-2/AM for 45 min at room temperature in HEPES buffer (137 mM NaCl, 2.7 mM KCl, 0.3 mM CaCl 2 , 1.0 mM MgCl 2 , 0.4 mM NaH 2 PO 4 , 5.6 mM glucose, 10 mM HEPES, and NaOH to pH 7.4).…”