Ca²⁺ influx in human T lymphocytes is induced independently of inositol phosphate production by mobilization of intracellular Ca²⁺ stores. A study with the Ca²⁺ endoplasmic reticulum‐ATPase inhibitor thapsigargin

Abstract

Thapsigargin (TG), a sesquiterpene lactone and non-phorbol 12-myristate 13-acetate tumor promoter, stimulates a rapid increase in intracellular free Ca2+ [( Ca2+]i) in human T lymphocytes clone P28. The [Ca2+]i response to TG is sustained in the presence of 1 mM extracellular Ca2+, while it becomes transient in Ca2(+)-free medium suggesting that TG activates both the release of Ca2+ from intracellular stores and the entry of Ca2+ from extracellular spaces. TG-induced Ca2+ influx is completely abolished after cell depolarization caused by increased extracellular concentrations of K+. The rise in [Ca2+]i stimulated by TG occurs in the absence of detectable production of inositol phosphates. Moreover, TG does not alter the early biochemical events of T cell activation triggered through the CD2 or the CD3 T cell antigens. Indeed, both inositol phosphate production and intracellular pH increase induced by specific monoclonal antibodies (mAb) remain unchanged after TG treatment. These data suggest that in human T lymphocytes TG releases Ca2+ from an intracellular pool by a mechanism which is independent of the phospholipase C metabolic pathway. Preincubation with TG of T cell clone P28 empties both the CD2 and the CD3-sensitive intracellular Ca2+ pool(s). Conversely, prestimulation of T cell clone P28 by CD3 or CD2-specific mAb inhibits the Ca2(+)-mobilizing effect of TG. Thus it appears that TG and CD2- or CD3-specific mAb mobilize Ca2+ from common Ca2+ pool(s). Taken together, these results demonstrate that Ca2+ influx in human T cells may be linked to mobilization of intracellular Ca2+ pools and by a mechanism independent of phosphoinositide hydrolysis. They further indicate that the release of intracellular Ca2+ pool(s) may play a major role in the opening of cell membrane Ca2+ channels observed during the CD2- or CD3-induced stimulation of human T lymphocytes.

Phosphatidylcholine-specific Phospholipase C Regulates Thapsigargin-induced Calcium Influx in Human Lymphocytes

“…Moreover, the involvement of phosphoinositidespecific phospholipases is unlikely, as no phosphatidylinositol breakdown was observed in lymphocytes treated with thapsigargin. The latter observation agrees with previous findings that depletion of intracellular calcium stores by thapsigargin is not accompanied by the hydrolysis of phosphatidylinositols (9,(37)(38)(39).…”

PAC1 Receptor Activation by PACAP-38 Mediates Ca2+ Release from a cAMP-dependent Pool in Human Fetal Adrenal Gland Chromaffin Cells

“…The second most important feature of the present data was that PACAP-responsive Ca 2ϩ pools were insensitive to TG. It has been reported that TG triggers the release of Ca 2ϩ from major nonmitochondrial Ca 2ϩ stores (46), including the IP 3 -sensitive Ca 2ϩ pool (47). Our data conclusively demonstrate that preincubation of the cells with TG or chronic application of high concentrations of TG (8 M) have no effect on the Ca 2ϩ response triggered by PACAP-38.…”

NAADP Mobilizes Calcium from the Endoplasmic Reticular Ca2+ Store in T-lymphocytes

“…Although the specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca 2ϩ ATPases (SERCA), thapsigargin (29), is an effective and specific tool to deplete ER/SRtype Ca 2ϩ stores, in intact, electrically non-excitable cells, there is often the problem of capacitative Ca 2ϩ entry being switched on immediately (30,31). Thus, to avoid any effects of capacitative Ca 2ϩ entry, all further experiments were conducted in the presence of GdCl 3 in the extracellular buffer.…”