Abstract
Elevation of extracellular Ca2+ concentration induces intracellular Ca2+ signaling in parathyroid cells. The response is due to stimulation of the phospholipase C/Ca2+ pathways, but the direct mechanism responsible for the rise of intracellular Ca2+ concentration has remained elusive. Here, we describe the electrophysiological property associated with intracellular Ca2+ signaling in frog parathyroid cells and show that Ca2+-activated Cl− channels are activated by intracellular Ca2+ increase through an inositol 1,4,5-trisphophate (IP3)-independent pathway. High extracellular Ca2+ induced an outwardly-rectifying conductance in a dose-dependent manner (EC50∼6 mM). The conductance was composed of an instantaneous time-independent component and a slowly activating time-dependent component and displayed a deactivating inward tail current. Extracellular Ca2+-induced and Ca2+ dialysis-induced currents reversed at the equilibrium potential of Cl− and were inhibited by niflumic acid (a specific blocker of Ca2+-activated Cl− channel). Gramicidin-perforated whole-cell recording displayed the shift of the reversal potential in extracellular Ca2+-induced current, suggesting the change of intracellular Cl− concentration in a few minutes. Extracellular Ca2+-induced currents displayed a moderate dependency on guanosine triphosphate (GTP). All blockers for phospholipase C, diacylglycerol (DAG) lipase, monoacylglycerol (MAG) lipase and lipoxygenase inhibited extracellular Ca2+-induced current. IP3 dialysis failed to induce conductance increase, but 2-arachidonoylglycerol (2-AG), arachidonic acid and 12S-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12(S)-HPETE) dialysis increased the conductance identical to extracellular Ca2+-induced conductance. These results indicate that high extracellular Ca2+ raises intracellular Ca2+ concentration through the DAG lipase/lipoxygenase pathway, resulting in the activation of Cl− conductance.
Highlights
Parathyroid hormone (PTH) regulates extracellular free Ca2+ concentration ([Ca2+]o) in cooperation with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3)and calcitonin
We report that frog parathyroid cells possess Ca2+-activated Cl2 channels and that these channels can be activated by an increase of [Ca2+]i through the mediation of the arachidonic acid cascade
Neither IP3 nor the DAG analog elicited any response in the phospholipase C pathway of frog parathyroid cells, we studied the following step in the phosphatidylinositol 4,5-diphosphate (PIP2) metabolism
Summary
Parathyroid hormone (PTH) regulates extracellular free Ca2+ concentration ([Ca2+]o) in cooperation with 1,25-dihydroxycholecalciferol (1,25-(OH)2D3)and calcitonin. On the other hand, [Ca2+]o regulates the secretion of PTH from parathyroid cells through an extracellular Ca2+-sensing receptor (CaR) [1,2]. High [Ca2+]o inhibits the secretion, whereas low [Ca2+]o enhances the secretion. It is believed that extracellular Ca2+ binds to CaR, and as a consequence inhibits the secretion of PTH via intracellular free Ca2+ concentration ([Ca2+]i). The molecular mechanism by which [Ca2+]i regulates the secretion is not well elucidated. The CaR belongs to the family C of G protein-coupled receptors (GPCRs) and has a large extracellular domain that binds external Ca2+ and other CaR agonists. Calcium binding to the receptor results in G protein-dependent activation of phosphatidylinositol-specific phospholipase C (PI-PLC) causing accumulation of inositol 1,4,5-
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