Shoji Sakano

Ca Mobilizing Action of Sphingosine in Jurkat Human Leukemia T Cells EVIDENCE THAT SPHINGOSINE RELEASES Ca FROM INOSITOL TRISPHOSPHATE- AND PHOSPHATIDIC ACID-SENSITIVE INTRACELLULAR STORES THROUGH A MECHANISM INDEPENDENT OF INOSITOL TRISPHOSPHATE

Effects of sphingosine on Ca mobilization in the human Jurkat T cell line were examined. Sphingosine increased the cytoplasmic Ca concentration ([Ca]) in a dose-dependent manner with an ED of around 8 μM. Sphingosine and OKT3, a CD3 monoclonal antibody, transiently increased [Ca], which declined to the resting level in the absence of extracellular Ca. Under the same conditions, pretreatment with sphingosine inhibited but did not abolish an increase in [Ca] induced by the subsequent addition of OKT3 and vice versa. However, pretreatment with sphingosine did not affect an increase in [Ca] induced by OKT3 in the presence of Ca. OKT3 increased IP formation, but sphingosine did not affect the level of IP by itself nor did it cause IP formation induced by OKT3. In permeabilized Jurkat cells, the addition of IP released Ca from nonmitochondrial intracellular stores, but the addition of sphingosine did not. Sphingosine, stearylamine, and psychosine increased [Ca] and diacylglycerol (DG) kinase activation; however, ceramide did not, whereas sphingosine 1-phosphate slightly activated DG kinase without elevation of [Ca]. Pretreatment with R59022, a DG kinase inhibitor, abolished the peak but did not affect the sustained response of [Ca] to sphingosine. Phosphatidic acid (PA) elevated [Ca], after which it declined to a resting level even in the presence of extracellular Ca. In accordance with this, PA did not stimulate Ca uptake into cells, but sphingosine and OKT3 did. Pretreatment with PA partially inhibited a rise in [Ca] induced by the subsequent addition of sphingosine and vice versa in the absence of extracellular Ca. Under similar conditions, pretreatment with PA affected an elevation of [Ca] induced by OKT3 less, after which the subsequent addition of sphingosine did not increase [Ca]. In permeabilized Jurkat cells, the addition of IP did not release Ca, but PA did in the presence of heparin. Pretreatment with thapsigargin, a microsomal Ca-ATPase inhibitor, abolished the rises of [Ca] induced by the subsequent addition of sphingosine, OKT3, and PA in the absence of extracellular Ca. The present results suggest that at least two kinds of intracellular Ca stores exist in Jurkat cells, both of which are IP- and PA-sensitive, and that sphingosine mobilizes Ca from both stores in an IP-independent manner. Furthermore, the IP- but not the PA-sensitive intracellular Ca store seems to regulate Ca entry induced by sphingosine.

Role of α1-adrenoceptor subtypes which mediate positive chronotropy in neonatal rat cardiac myocytes

We investigated the involvement of alpha 1-adrenoceptor subtypes in the positive chronotropic response to norepinephrine (NE) in neonatal rat cardiac myocytes at day 3 of culture. The cardiac myocytes at day 3 of culture exhibited a dose-dependent positive chronotropic response to NE in the presence of propranolol, a beta-adrenoceptor antagonist. The positive chronotropic responses to NE were completely antagonized by the alpha 1-adrenoceptor antagonist prazosin. The NE-induced positive chronotropic response was inhibited 68% by the alpha 1B-adrenoceptor antagonist, chloroethylclonidine (CEC), but partially (41%) so by the alpha 1A-adrenoceptor antagonist, WB4101. In the membrane fraction derived from cardiac myocytes at day 3 of culture, pretreatment with CEC decreased the Bmax of the alpha 1-adrenoceptor to 22% of the control value. The NE-induced positive chronotropic response was inhibited 62 and 77% by the voltage-gated Ca2+ channel blocker such as nifedipine and verapamil, respectively. These findings indicate (1) that cultured neonatal rat cardiac myocytes possess both alpha 1-adrenoceptor subtypes, i.e., alpha 1A and alpha 1B, (2) that the predominant alpha 1-adrenoceptor subtypes mediating NE-induced positive chronotropy in neonatal rat cardiac myocytes at day 3 of culture are alpha 1B-subtypes, and (3) that NE-induced positive chronotropy may be caused via voltage-gated Ca2+ channel activation.

Inhibitory effects of tyrosine kinase inhibitors on capacitative Ca2+ entry in rat glioma C6 cells.

The effects of genistein and erbstatin analogue, inhibitors of tyrosine kinase, on Ca2+ mobilization evoked by thapsigargin (TG) were examined in rat glioma C6 cells. Genistein and erbstatin analogue inhibited the Ca2+ release from intracellular pools as well as Ca2+ entry from extracellular medium evoked by TG in a dose-dependent manner. However, they did not affect a Ca2+ entry due to leakage of Ca2+ from extracellular medium into cells. The present results suggest that tyrosine kinase inhibitors inhibit capacitative Ca2+ entry due to the inhibition of both Ca2+ entry itself and Ca2+ release in rat glioma C6 cells.