Identification of intracellular calcium pools and calcium translocation mechanisms in DDT(1)MF-2 smooth muscle cells

Abstract

The focus of the present studies is on the functional characterization of discrete Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools on the basis of subcellular location, Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport properties, and functional roles. In both saponin-permeabilized DDT{dollar}\sb1{dollar}MF-2 smooth muscle cells and purified rough endoplasmic reticulum (ER) vesicles from these cells, sphingosine derivatives, in particular, sphingosine (Sph) and sphingosyl-phosphorylcholine (SPC) were observed to induce rapid and profound intracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar} release as revealed by {dollar}\sp{lcub}45{rcub}{dollar}Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake and release assays. The properties of sphingoid base-induced Ca{dollar}\sp{lcub}2+{rcub}{dollar} release were found highly comparable to their reported effects on PKC inhibition in terms of sensitivity, molecular specificity and reversibility. Sphingoid bases released Ca{dollar}\sp{lcub}2+{rcub}{dollar} up to the same level of the combined action of inositol-1,4,5-triphosphate (InsP{dollar}\sb3){dollar} and GTP, and no further Ca{dollar}\sp{lcub}2+{rcub}{dollar} release induced by InsP{dollar}\sb3{dollar} together with GTP was seen after maximal effect of sphingosine derivatives, indicating a shared Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool target with InsP{dollar}\sb3{dollar} and GTP. The action of Sph, in contrast to that of SPC, was blocked by ADP (100 {dollar}\mu{dollar}M) addition, ATP depletion and low temperature, indicating that Sph had no intrinsic activity in releasing Ca{dollar}\sp{lcub}2+{rcub}{dollar} and must be converted into an active form. The results indicate that generation of sphingoid bases in cells may activate a dual signaling pathway involving Ca{dollar}\sp{lcub}2+{rcub}{dollar} release in parallel to PKC inhibition. Thapsigargin (TG) inhibited 75% of total intracellular ATP-dependent Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake with an IC{dollar}\sb{lcub}50{rcub}{dollar} of 30 nM. TG-sensitive Ca{dollar}\sp{lcub}2+{rcub}{dollar} pumps exist in two previously defined pools--the InsP{dollar}\sb3{dollar}-sensitive, oxalate-permeable Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool, and the InsP{dollar}\sb3{dollar}-insensitive, oxalate-impermeable Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool. A brief TG treatment of 30 min caused persistent Ca{dollar}\sp{lcub}2+{rcub}{dollar} depletion in TG-sensitive pools as well as total arrest of cell growth. A precise correlation between the Ca{dollar}\sp{lcub}2+{rcub}{dollar} content in signaling pools and cell growth was shown to exist. (Abstract shortened by UMI.)