Fatty acids with chain lengths of more than 12 carbon atoms stimulate cholecystokinin (CCK) release from enteroendocrine cells in vivo (McLaughlin et al. 1999) and in vitro, accompanied with an increase in [Ca²⁺]_i (McLaughlin et al. 1998). By monitoring [Ca²⁺]_i in an established murine CCK-producing enteroendocrine cell line STC-1, we investigated which signal pathways transduce the fatty acid signal, or whether fatty acids themselves act intracellularly to induce a Ca²⁺ signal, and hence secretion.

STC-1 cells were loaded with Ca²⁺-sensitive fluorescent dyes and [Ca²⁺]_i measured ratiometrically, using a fluorescence-microscope imaging system. Intact cells loaded with fura-2 were exposed to 100-500 µM fatty acid (C8:0, C10:0, C12:0, C18:1) under several conditions, e.g. absence of Ca²⁺₀, or pretreatment with drugs known to block candidate signal transduction pathways. To examine direct effects of fatty acids on the intracellular Ca²⁺ store, Ca²⁺ release was assessed by measuring intra-organellar Ca²⁺ in cells loaded with mag-fura-2 (a low affinity Ca²⁺-sensitive dye) and permeabilized by Streptolysin O (van de Put & Elliott, 1996).

In intact cells, in the presence or absence of extracellular Ca²⁺, C12:0 and C18:1, but not C8:0 or C10:0, induced [Ca²⁺]_i responses in a dose-dependent manner. The C12:0-induced [Ca²⁺]_i response was prevented by depletion of intracellular Ca²⁺ stores with thapsigargin. Several blockers of classical signal pathways, which may couple to Ca²⁺ release from intracellular stores (IP₃ receptor antagonists, ryanodine receptor antagonists, phospholipase inhibitors and cAMP), all failed to abolish C12:0-induced [Ca²⁺]_i responses.

In permeabilized cells, Ca²⁺ was accumulated by stores in the presence of ATP, and was released by IP₃ or thapsigargin. C12:0 (100~500 µM) released stored Ca²⁺ in a dose-dependent manner. The fatty acid chain length dependency was identical in permeabilized cells and intact cells.

Fatty acids (C12:0 and C18:1) induce Ca²⁺ release from intracellular Ca²⁺ stores, and this can occur in the absence of extracellular Ca²⁺. The data strongly suggest that these fatty acids can act directly on an intracellular Ca²⁺ store to release Ca²⁺, independently of the major intracellular signal pathways.

This work was supported by the Digestive Disorders Foundation Senior Fellowship (JMcL), and the Japan Society for the Promotion of Science (TH).