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Evidence That Calcium Entry Into Calcium-Transporting Dental Enamel Cells Is Regulated by Cholecystokinin, Acetylcholine and ATP.

ABSTRACT: Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca²⁺ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca²⁺ safely remain unclear. Our previous work contradicted the dogma that Ca²⁺ is ferried through the cytosol of Ca²⁺-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca²⁺ stores and their concomitant refilling by store-operated Ca²⁺ entry (SOCE) mediated by Ca²⁺ release activated Ca²⁺ (CRAC) channels. Given that Ca²⁺ handling is maximal during the enamel-mineralization stage (maturation), we anticipated that SOCE would also be elevated then. Confirmation was obtained here using single-cell recordings of cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in rat ameloblasts. A candidate SOCE agonist, cholecystokinin (CCK), was found to be upregulated during maturation, with Cck transcript abundance reaching 30% of that in brain. CCK-receptor transcripts were also detected and Ca²⁺ imaging showed that CCK stimulation increased [Ca²⁺]_cyt in a dose-responsive manner that was sensitive to CRAC-channel inhibitors. Similar effects were observed with two other SOCE activators, acetylcholine and ATP, whose receptors were also found in enamel cells. These results provide the first evidence of a potential regulatory system for SOCE in enamel cells and so strengthen the Ca²⁺ transcytosis paradigm for ER-based transport of bulk Ca²⁺. Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca²⁺ transport.

SUBMITTER: Nurbaeva MK

PROVIDER: S-EPMC6036146 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Evidence That Calcium Entry Into Calcium-Transporting Dental Enamel Cells Is Regulated by Cholecystokinin, Acetylcholine and ATP.

Nurbaeva Meerim K MK Eckstein Miriam M Devotta Arun A Saint-Jeannet Jean-Pierre JP Yule David I DI Hubbard Michael J MJ Lacruz Rodrigo S RS

Frontiers in physiology 20180702

Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca2+ while producing the most highly mineralized tissue. However, the mechanisms used by enamel cells to handle bulk Ca2+ safely remain unclear. Our previous work contradicted the dogma that Ca2+ is ferried through the cytosol of Ca2+-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticu ...[more]

PMID: 30013487

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Project description:BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.Methodology/principal findingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.Conclusions/significanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

Dataset Information

Evidence That Calcium Entry Into Calcium-Transporting Dental Enamel Cells Is Regulated by Cholecystokinin, Acetylcholine and ATP.

Publications

Evidence That Calcium Entry Into Calcium-Transporting Dental Enamel Cells Is Regulated by Cholecystokinin, Acetylcholine and ATP.

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