• Modification of calcium pool function by fatty acids and their coenzyme A esters

      Rys-Sikora, Krystyna Ewa; Gill, Donald L. (1997)
      Intracellular Ca2+ pools are essential elements in the generation of Ca2+ signals within cells, however, their nature and identity have remained elusive. Ca2+ pools are complex and dynamic entities and are modified by G protein-induced membrane fusion events, allowing GTP-activated transfer of Ca2+ between discrete Ca2+ pools. Studies examined the modification of GTP-activated Ca2+ translocation process by fatty acyl-CoA esters and fatty acids since G protein action can be modified by fatty acylation. Using permeabilized DDT1MF-2 smooth muscle cells, palmitoyl-CoA (IC50 = 0.5 muM) was observed to completely block 45Ca2+ release activated by GTP, while having no effect on InsP3-induced Ca2+ release. Fatty acyl chain length was important, only C-13 to C-16 fatty acyl-CoA esters fully inhibited the action of GTP. CoA(10 muM) also blocked GTP-activated Ca2+ release, although the free sulfhydryl group and ATP requirements indicated that CoA must be fatty acylated to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA. Thus, fatty acyl transfer is not required indicating that the blockade is due to a direct allosteric modification of a component of the GTP-activated process. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release. In the presence of oxalate, GTP-activated Ca2+ transfer causes a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect. These results provide strong evidence that GTP-activated Ca2+ translocation does not reflect a full fusion event, but the formation of a reversible prefusion pore. The actions of fatty acids were very different from their acyl-CoA esters; 10-100 muM palmitate (C16:0) had a major stimulatory effect on GTP-mediated Ca2+accumulation. The biphasic nature of the palmitate effect was characteristically similar to the effect of oxalate; however, the EC50 for palmitate was 20 muM (approx. 100-fold lower that of oxalate). This activation was highly specific for chain length and degree of saturation. Only pentadecanoic acid (C15:0) duplicated this effect, unsaturated fatty acids were completely ineffective. Both palmitate- and oxalate-activated Ca2+ accumulation in the presence of GTP were inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). Hence, both Ca2+-complexing agents may enter anion-permeable Ca2+ subpools through similar anion channels. To further examine the nature of these Ca2+ complexes, a comparison of the releasability of Ca2+ using InsP3 and the Ca2+ ionophore, A23187 was undertaken. In the presence of oxalate, GTP-mediated accumulation of Ca2+ was only slowly releasable by InsP3 or A23187. Whereas, Ca2+ accumulated in the presence of palmitate and GTP was completely releasable by A23187, only a small fraction of the accumulated Ca2+was released by InsP3. These data suggest important differences between the state and possibly, the location of oxalate- and palmitate-Ca2+ complexes within Ca2+ pools. Thus, the formation of Ca2+-fatty acid complexes and, in turn, the activation of Ca2+ accumulation may reflect a major physiological role for fatty acids in stabilizing Ca2+ within the lumen of Ca2+ pools.