Microtubules regulate local Ca2+ spiking in secretory epithelial cells

The role of the cytoskeleton in regulating Ca2+ release has been explored in epithelial cells. Trains of local Ca2+ spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca2+-dependent Cl- current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments, In contrast, nocodazole (5-100 mu M), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me2SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca2+ signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca2+ response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca2+ spikes at least in part because of the local positioning of the endoplasmic reticulum.