NMDA receptors as a possible component of store-operated Ca super(2+) entry in human T-lymphocytes

Elevation of intracellular Ca super(2+) in T-lymphocytes as a consequence of T cell antigen receptor activation triggers transcriptional programs resulting in effector cytokine secretion and immune response coordination. Increase of Ca super(2+) concentration in T-lymphocytes follows both the Ins(1,4,5)P sub(3)-dependent release from an intracellular store and subsequent influx from extracellular milieu. Flow cytometry and the fluorescent dye Fluo-4AM have been used to demonstrate that noncompetitive NMDA receptor antagonist (+)-MK801 inhibits Ca super(2+) influx in T cells induced by thapsigargin. Combination of thapsigargin and (+)-MK801 with following incubation does not affect Ca super(2+) mobilization from intracellular stores, while decreased Ca super(2+) entry was observed. Overall data indicate that the ion channel blocker (+)-MK801 is able to inhibit the Ca super(2+) influx and confirm our suggestion about involvement of NMDA receptor in the store-operated Ca super(2+) entry mechanisms in human T-lymphocytes. To identify the signal transduction pathways associated with NMDA receptors in mitogen-stimulated T-lymphocytes, the cells were incubated with (+)-MK801, then activity of key phosphorylated protein kinases of MAP-activated (pERK1/2, pSAPK/JNK, p-p38), Ca super(2+)-dependent (pCaMKII), PI3/Akt-dependent (pGSK-3 beta ), and PKC-activated (pPKC[thetas]) pathways were detected. The data we obtained demonstrate that (+)-MK801 treatment leads to more prominent decrease in Ras-activated protein kinases pERK1/2 and Rac-activated proteins p-p38 and pSAPK/JNK, as compared to DAG-dependent pPKC[thetas] and Ca super(2+)-dependent pCaMKII. These results show that NMDA receptors are mainly involved in regulation of Ras/Rac-dependent signaling in T-lymphocytes.