Takahiko Kumada

Antigen-mediated phospholipase D activation in rat basophilic leukemia (RBL-2H3) cells: possible involvement of calcium/calmodulin.

The differential implication of protein kinase C (PKC) isozymes in antigen- or PMA-induced phospholipase D (PLD) activation was investigated in rat basophilic leukemia (RBL-2H3) cells. In [3H]oleic acid-labeled cells, both antigen (100 ng/ml) and phorbol 12-myristate 13-acetate (PMA) (100 nM) produced a specific product of PLD activation, [3H]phosphatidylbutanol (PBut) in the presence of butanol. Pretreatment of cells with a selective PKC inhibitor, Ro31-8425 (1-5 microM) inhibited PMA-stimulated PLD activity by 85%. In contrast, the antigen-stimulated PLD activity was much less sensitive to the inhibitor. RBL-2H3 cells express PKC alpha, beta, delta, epsilon and zeta isozymes and down-regulation of PKC by exposure to PMA (20 nM) for 1-2 h caused rapid decrease in PKC alpha and beta isozymes, leaving PKC delta, epsilon and zeta isozymes intact. Apparent decreases in the levels of PKC alpha and beta to about 50% were observed after adding 20 nM PMA for 1 h, when PMA-stimulated PLD activity was inhibited by up to 70%. Decrease in antigen-stimulated PLD activity was evident after 2 h PMA-treatment, when PKC alpha and beta decreased by nearly 70%. These results suggest that in the antigen-mediated PLD pathway PKC may be implicated but not play such a great role as PMA-stimulated pathway which is mediated through PKC alpha or beta. Then, we have examined the involvement of calcium/calmodulin (CaM) in PLD activation by antigen, since the antigen-stimulated PLD activation showed the absolute requirement for extracellular calcium. Preincubation of RBL-2H3 cells with a CaM antagonist W-7 (20 microM) inhibited the antigen-stimulated PLD activity by 90%, but W-5, a chlorine-deficient analogue of W-7 that only weakly interact with CaM, caused little inhibitory effect. Another non-specific CaM antagonist, trifluoperazine (TFP) also inhibited PLD activation. These results suggest that calcium/CaM may be involved in the antigen-stimulated PLD activation.

Brefeldin A Inhibits Antigen- or Calcium Ionophore-Mediated but not PMA-Induced Phospholipase D Activation in Rat Basophilic Leukemia (RBL-21-13) Cells

Recent reports have indicated that ADP-ribosylation factor (ARF) plays a role in the regulation of phospholipase D (PLD) activity in the in vitro assay system. Since a fungal metabolite brefeldin A (BFA) is known to interfere with ARF function, the effect of BFA on antigen-induced PLD activation was examined in rat basophilic leukemia (RBL-2H3) cells. BFA inhibited the antigen-induced formation of phosphatidylbutanol (PBut), a specific and stable metabolite produced by PLD activity in a concentration-dependent manner. The maximal inhibition obtained at 10 micrograms/ml of the drug was nearly 70% and further inhibition was not observed at higher concentrations. Ca(2+)-ionophore A23187-mediated PLD activation was also prevented by BFA. In contrast, BFA failed to inhibit PLD activation in response to 4 beta-phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC). This indicates that there are BFA-sensitive and BFA-insensitive pathways leading to PLD activation in RBL-2H3 cells and also that the PKC-mediated pathway may be insensitive to BFA treatment, suggesting the existence of PLD isozymes. BFA inhibited Ag-induced serotonin release at a concentration 20-fold lower than that needed for the inhibition of PLD. Moreover, PMA caused a marked production of PBut, but it failed to elicit secretory response. This implies that PLD may be not a crucial element for secretory responses.

Phenylarsine Oxide (PAO)-Mediated Activation of Phospholipase D in Rat Basophilic Leukemia (RBL-2H3) Cells: Possible Involvement of Calcium and Protein Kinase C

Addition of phenylarsine oxide (PAO) to [3H]oleic acid-labeled rat basophilic leukemia (RBL-2H3) cells gave rise to the remarkable formation of [3H]phosphatidylbutanol (PBut), a specific product of phospholipase D (PLD) activation. Preincubation of cells with 2,3-dimercaptopropanol (DMP) or dithiothreitol (DTT), compounds containing sulfhydryls, prevented PAO-stimulated [3H]PBut formation, indicating that PAO-stimulated PLD through interacting with vicinal thiol groups. Treatment of cells with PAO resulted in increase in intracellular Ca2+ concentration without significant production of inositol phosphates. Removal of extracellular free Ca2+ by chelating with EGTA was found to inhibit [3H]PBut formation by PAO. Incubation of cells with 20 nM phorbol 12-myristate 13-acetate (PMA) for 6 h caused down-regulation of protein kinase C (PKC) alpha and beta isozymes, whereas it had no effect on PKC delta, epsilon and zeta isozymes. Under this condition, decrease in PAO-stimulated [3H]PBut formation was observed to occur with a concomitant decrease in the level of PKC alpha and beta isozymes. These results suggest that a covalent bridge between vicinal thiol groups of cell surface proteins induced by PAO potentiates PLD activation and that PAO-induced PLD activation is regulated by Ca2+ and PKC alpha and/or beta isozymes.

Evaluation of Treatment of Vestibular Vertigo. Evaluation of Objective Findings.

We report our assessment of objective findings as a method of evaluation of the effect of treatment of vestibular vertigo (Gifu University method). The following should be investigated, (1) effect of treatment on vertigo, (2) effect of treatment on damage to the inner ear, (3) effect of treatment on recurrent attacks of vertigo, and (4) objective effect of treatment on improvement of activities of daily living.We reviewed the period of evaluation after treatment of recurrent attacks of vertigo, and formulated a numeric equation for the number of cluster attacks.The effect of treatment on recurrent attacks of vertigo was evaluated by comparing the number of attacks a month after the start of treatment with the number before treatment.Twenty-six patients with Menieres disease were questioned about the number of attacks of vertigo and the effect of treatment one month, 6 months, 12 months and 24 months after the start of treatment. The results indicated improvement after one month of treatment in 11 patients (42%), after 6 months in 16 patients (62%), and after 12 months in 19 patients (73%). After 24 months of treatment, the figures were unchanged. A period of at least 12 months is necessary to evaluate the effect of treatment on recurrent attacks of vertigo.In patients with cluster attacks of vertigo, the effect of treatment must be evaluated differently. The duration of a cluster and the number of attacks in a cluster must be compared before and after treatment.