Acidic Calcium Stores Contribute to Secretory Activity Following Elevation of Camp in the Salivary Gland

Abstract

Calcium signaling in response to autonomic neural input drives fluid and protein secretion within salivary glands. Previous research focused primarily on the canonical IP3-mediated calcium release from ER stores. However, other important reservoirs for calcium release exist such as lysosomes, endosomes, and secretory granules. Whether and how these acidic stores are activated remains largely undefined in terms of their contributions to global calcium signals, protein exocytosis and saliva production in salivary tissue. To address this question we used lysotracker red, a fluorescent dye that labels acidic organelles. Confocal images demonstrated that mouse parotid acini had abundant stores that were apically located and whose pH gradients were dissipated by treatment with the vacuolar H-ATPase inhibitor bafilomycin A1. In contrast, parotid ductal cells displayed only a punctate and dispersed or perinuclear labeling. The functional effect of bafilomycin treatment was tested using live cell imaging methods. Bafilomycin treatment resulted in a two-thirds diminishment of peak calcium release amplitudes evoked by a threshold level of carbachol stimulation. The diminishment in peak amplitude and in the rate of rise of the evoked calcium signal was only evident in cells where cAMP was first elevated by either forskolin or the beta-adrenergic agonist isoproterenol. There is evidence that acidic store calcium release can be triggered by the dinucleotide second messenger NAADP. Consistent with this, NED19, a NAADP receptor antagonist, diminished by half the peak calcium response. This data indicates that calcium release from acidic stores in mouse parotid acinar cells is cAMP-dependent and may be mediated by NAADP. In addition, bafilomycin and NED19 treatment significantly reduced exocytotic secretory activity, even without cAMP elevation, supporting the idea that acidic stores may contribute to vesicle fusion and protein release.