Dimethylsphingosine increases cytosolic calcium and intracellular pH in human T lymphocytes

Abstract

N,N-Dimethyl- d-erythro-sphingosine (DMS) is the N-methyl derivative of sphingosine; both are activators of sphingosine-dependent protein kinases. The aim of this work was to study the effect of DMS on cytosolic calcium and intracellular pH (pHi) in human T lymphocytes. The variations of calcium and pH were determined by fluorescence digital imaging using Fura-2-AM and BCECF-AM, respectively. DMS increased both pHi and Ca 2+-cytoslic in human T lymphocytes. These effects were dose-dependent. This drug induced a fast increase in pHi and a release of calcium from different intracellular calcium pools than thapsigargin. DMS also induced a Ca 2+-influx different from the store-operated calcium channels, since drug effect was not modified by 30 μM SKF 96365. The influx of calcium induced by DMS was completely blocked by preincubation in the presence of nickel, or lanthanum, while the increase in pHi was no affected. However, the presence of cadmium reduced but does not block Ca 2+-influx. The inhibition of G-protein by 100 ng/mL pertussis toxin, and the inhibition of tyrosine kinases by genistein significantly reduced the cytosolic calcium increase induced by DMS by an inhibition of both, release of calcium from intracellular pools and influx from extracellular medium. The inhibition of pools emptiness by these drugs was related with the inhibition that they induce in the DMS cytosolic alcalinization. In summary, DMS increases pHi and as consequence releases calcium from intracellular pools, and it increases calcium-influx through a channel different from store-operated channel (SOC). Both cytosolic calcium and pHi increase are modulated by G-proteins and tyrosine kinases.