Intestinal enteroendocrine cells rely on ryanodine and IP₃ calcium store receptors for mechanotransduction

Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5-HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na⁺ , K⁺ , Ca²⁺ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca²⁺ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca²⁺ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP-1) resulted in a rapid increase in cytoplasmic Ca²⁺ and a slower decrease in ER stores Ca²⁺ , suggesting the involvement of intracellular Ca²⁺ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca²⁺ store receptors, a similar expression of IP₃ receptors, but a >30-fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca²⁺ responses decreased dramatically by emptying stores and pharmacologically blocking IP₃ and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca²⁺ responses and 5-HT release. In tissue, pressure-induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca²⁺ stores to amplify mechanically induced Ca²⁺ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca²⁺ signalling, 5-HT release and epithelial secretion. KEY POINTS: A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca²⁺ ) with a longer duration than the stimulus, suggesting intracellular Ca²⁺ signal amplification. In this study, we profiled the expression of intracellular Ca²⁺ store receptors and found an enriched expression of the intracellular Ca²⁺ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5-HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca²⁺ stores and are selective in their use of Ryr3 for amplification of intracellular Ca²⁺ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.