RESUMO
BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.
RESUMO
The importance of the immediately releasable pool (IRP) of vesicles was proposed to reside in the maintenance of chromaffin cell secretion during the firing of action potentials at basal physiological frequencies. To accomplish this duty, IRP should be replenished as a function of time. We have previously reported that an action potential-like stimulus (APls) triggers the release of ~50% IRP, followed by a fast dynamin-dependent endocytosis and an associated rapid replenishment process. In this work, we investigated the endocytosis and IRP replenishment produced after the exocytosis of variable IRP fractions in mice primary chromaffin cell cultures. Exocytosis and endocytosis were estimated by membrane capacitance measurements obtained in patch-clamped cells. In addition to the dynamin-dependent fast endocytosis activated after the application of APls or 5 ms squared depolarizations, we found that depolarizations lasting 25-50 ms, which release >80% of IRP, are related with a fast dynamin-independent, Ca2+ - and protein kinase C (PKC)-dependent endocytosis (time constant <1 s). PKC inhibitors, such as staurosporine, bisindolylmaleimide XI, PKC 19-31 peptide, and prolonged treatments with high concentrations of phorbol esters, reduced and decelerated this endocytosis. Additionally, we found that the inhibition of PKC also abolished a slow component of replenishment (time constant ~8 s) observed after total IRP exocytosis. Therefore, our results suggest that PKC contributes to the coordination of membrane retrieval and vesicle replenishment mechanisms that occur after the complete exocytosis of IRP.
