inositol-1-4-5-trisphosphate and Lymphoma--B-Cell

A malachite green-conjugated inositol 1,4,5-trisphosphate (MGIP(3)) induces specific inactivation of IP(3) receptor (IP(3)R) in tissue samples upon laser irradiation. To verify potential usefulness of the method for studies of cellular Ca(2+) signaling, we conducted laser inactivation at the single-cell level and show that IP(3)R was inactivated with extremely high spatiotemporal resolution. In the presence of MGIP(3), the Ca(2+) release function of IP(3)R in single B lymphoma cells decayed exponentially with increasing duration of laser irradiation with a time constant of 3.4 s. Moreover, by confining laser irradiation to a spatially distinct region of differentiated PC12 cells, subcellular inactivation of IP(3)R was attained, as revealed by a loss of local Ca(2+) signal. Such real-time inactivation of IP(3)R only within a subcellular region may provide a powerful method for investigating spatiotemporal dynamics of Ca(2+) signaling.

Topics: Animals; Calcium Channels; Calcium Signaling; Chickens; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ion Channel Gating; Lasers; Lymphoma, B-Cell; Methods; Molecular Probes; PC12 Cells; Rats; Receptors, Cytoplasmic and Nuclear; Rosaniline Dyes; Time Factors

We examined the effects of low energy electromagnetic field (EMF) exposure on the BTK kinase activity in B18-2 ([Btk-, rBTK(wt)] DT40) chicken lymphoma B cells and NALM-6 leukemic pre-B cells. Exposure of B 18-2 cells to EMF resulted in activation of BTK within 1 to 15 minutes in 8 of 8 independent experiments with stimulation indexes ranging from 1.2 to 13.3. While in some experiments the BTK stimulation was transient, in others the BTK activity continued to be significantly elevated for up to 4 hours. Similarly, exposure of NALM-6 cells to EMF resulted in activation of BTK within 30 minutes in 7 of 7 experiments with stimulation indexes ranging from 1.2 to 7.4. Stimulation of BTK activity in EMF exposed cells was associated with enhanced phosphoinositide turnover and increased inositol-1,4,5-trisphosphate (IP3) production in 7 of 13 experiments with DT40 cells and 7 of 13 experiments with NALM-6 cells. The likelihood and magnitude of an IP3 response after EMF exposure were similar to those after BCR ligation on DT40 cells and CD19 ligation on NALM-6 cells. These results confirm and extend our previous studies regarding EMF-induced biochemical signaling events in B-lineage lymphoid cells.

Topics: Agammaglobulinaemia Tyrosine Kinase; Animals; Chickens; Electromagnetic Fields; Enzyme Activation; Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Kinetics; Leukemia, B-Cell; Lymphoma, B-Cell; Phospholipase C gamma; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Type C Phospholipases

Exposure of wild-type DT40 lymphoma B cells or Bruton's tyrosine kinase (BTK)-deficient DT40 cells reconstituted with the human btk gene to a 1-gauss 60-Hz electromagnetic field (EMF) has been reported to rapidly increase inositol 1,4,5-trisphosphate (Ins 1,4, 5-P3) production (1,2). Here we have used BTK-deficient DT40 B cells reconstituted with the human btk gene to evaluate the reproducibility of these findings. An experimental design with blinded exposures and anti-IgM treatment to induce Ins 1,4,5-P3 production as a positive control, showed no significant effect of a 1-gauss 60-Hz EMF on Ins 1,4,5-P3 production. Because recent work has shown that the activation of BTK was required for EMF-responsiveness (2), we also evaluated the reproducibility of this finding in wild-type DT40 cells. BTK was activated in a dose- and time-dependent manner by treatment with the tyrosine phosphatase inhibitor pervanadate. However, the ability to detect BTK activation, as measured by increased autophosphorylation by immune complex kinase assay, was dependent on the kinase buffer. Using cells from the original investigators, no evidence was obtained to support the hypothesis that exposure to a 1-gauss 60-Hz EMF had a causal effect on protein-tyrosine kinase activities affecting Ins 1,4,5-P3 production.

Topics: Agammaglobulinaemia Tyrosine Kinase; Antibodies; Electromagnetic Fields; Enzyme Activation; Enzyme Induction; Humans; Immunoglobulin M; Inositol 1,4,5-Trisphosphate; Lymphoma, B-Cell; Phosphorylation; Protein-Tyrosine Kinases; Tumor Cells, Cultured; Vanadates