Novel Purine Derivative ITH15004 Facilitates Exocytosis through a Mitochondrial Calcium-Mediated Mechanism

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca2+ microdomain (HCMD) generated by Ca2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na+/Ca2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K+-depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca2+ concentrations ([Ca2+]c) K+-challenged BCCs, and (v) non-inactivated [Ca2+]c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca2+ handling during K+ depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

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Hypoxia-elicited catecholamine release is controlled by L-type as well as N/PQ types of calcium channels in rat embryo chromaffin cells

AJP Cell Physiology ◽

10.1152/ajpcell.00101.2014 ◽

2014 ◽

Vol 307 (5) ◽

pp. C455-C465 ◽

Cited By ~ 9

Author(s):

José-Carlos Fernández-Morales ◽

Juan-Fernando Padín ◽

Juan-Alberto Arranz-Tagarro ◽

Stefan Vestring ◽

Antonio G. García ◽

...

Keyword(s):

Calcium Channels ◽

Chromaffin Cells ◽

Relative Proportion ◽

Rat Embryo ◽

Hypoxia Exposure ◽

Hypoxic Conditions ◽

Relative Contribution ◽

Adrenal Chromaffin ◽

Bulk Response

At early life, the adrenal chromaffin cells respond with a catecholamine surge under hypoxic conditions. This response depends on Ca2+ entry through voltage-activated calcium channels (VACCs). We have investigated here three unresolved questions that concern this response in rat embryo chromaffin cells (ECCs): 1) the relative contribution of L (α1D, Cav1.3), N (α1B, Cav2.2), and PQ (α1A, Cav2.1) to the whole cell Ca2+ current ( ICa); 2) the relative contribution of L and N/PQ channels to the cytosolic Ca2+ elevations triggered by hypoxia (Δ[Ca2+]c); and 3) the role of L and non-L high-VACCs in the regulation of the catecholamine surge occurring during prolonged (1 min) hypoxia exposure of ECCs. Nimodipine halved peak ICa and blocked 60% the total Ca2+ entry during a 50-ms depolarizing pulse to 0 mV (QCa). Combined ω-agatoxin IVA plus ω-conotoxin GVIA (Aga/GVIA) blocked 30% of both ICa peak and QCa. This relative proportion of L- and non-L VACCs was corroborated by Western blot that indicated 55, 23, and 25% relative expression of L, N, and PQ VACCs. Exposure of ECCs to hypoxia elicited a mild but sustained Δ[Ca2+]c; the area of Δ[Ca2+]c was blocked 50% by nifedipine and 10% by Aga/GVIA. Exposure of ECCs to 1-min hypoxia elicited an initial transient burst of amperometric secretory spikes followed by scattered spikes along the time of cell exposure to hypoxia. This bulk response was blocked 85% by nimodipine and 35% by Aga/GVIA. Histograms on secretory spike frequency vs. time indicated a faster initial inactivation when Ca2+ entry took place through N/PQ channels; more sustained secretion but at a lower rate was associated to Ca2+ entry through L channels. The results suggest that the HIS response may initially be controlled by L and P/Q channels, but later on, N/PQ channels inactivate and the delayed HIS response is maintained at lower rate by slow-inactivating L channels.

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The role of nicotinic receptors and calcium channels in mipafox induced inhibition of catecholamine release in bovine chromaffin cells

Environmental Toxicology and Pharmacology ◽

10.1016/1382-6689(96)00017-8 ◽

1996 ◽

Vol 1 (4) ◽

pp. 241-247 ◽

Cited By ~ 4

Author(s):

Luis M. Gutiérrez ◽

Miguel A. Sogorb ◽

Eugenio Vilanova ◽

Salvador Viniegra

Keyword(s):

Calcium Channels ◽

Chromaffin Cells ◽

Nicotinic Receptors ◽

Catecholamine Release ◽

Bovine Chromaffin Cells

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Ca2+-induced Ca2+ Release in Chromaffin Cells Seen from inside the ER with Targeted Aequorin

The Journal of Cell Biology ◽

10.1083/jcb.144.2.241 ◽

1999 ◽

Vol 144 (2) ◽

pp. 241-254 ◽

Cited By ~ 134

Author(s):

Maria Teresa Alonso ◽

Maria José Barrero ◽

Pedro Michelena ◽

Estela Carnicero ◽

Inmaculada Cuchillo ◽

...

Keyword(s):

Chromaffin Cells ◽

Physiological Role ◽

Dependent Manner ◽

Excitable Cells ◽

Permeabilized Cells ◽

Low Concentrations ◽

Cyclic Adp Ribose ◽

Cell Depolarization ◽

Adrenal Chromaffin

The presence and physiological role of Ca2+-induced Ca2+ release (CICR) in nonmuscle excitable cells has been investigated only indirectly through measurements of cytosolic [Ca2+] ([Ca2+]c). Using targeted aequorin, we have directly monitored [Ca2+] changes inside the ER ([Ca2+]ER) in bovine adrenal chromaffin cells. Ca2+ entry induced by cell depolarization triggered a transient Ca2+ release from the ER that was highly dependent on [Ca2+]ER and sensitized by low concentrations of caffeine. Caffeine-induced Ca2+ release was quantal in nature due to modulation by [Ca2+]ER. Whereas caffeine released essentially all the Ca2+ from the ER, inositol 1,4,5-trisphosphate (InsP3)- producing agonists released only 60–80%. Both InsP3 and caffeine emptied completely the ER in digitonin-permeabilized cells whereas cyclic ADP-ribose had no effect. Ryanodine induced permanent emptying of the Ca2+ stores in a use-dependent manner after activation by caffeine. Fast confocal [Ca2+]c measurements showed that the wave of [Ca2+]c induced by 100-ms depolarizing pulses in voltage-clamped cells was delayed and reduced in intensity in ryanodine-treated cells. Our results indicate that the ER of chromaffin cells behaves mostly as a single homogeneous thapsigargin-sensitive Ca2+ pool that can release Ca2+ both via InsP3 receptors or CICR.

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Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells

Physiological Reviews ◽

10.1152/physrev.00039.2005 ◽

2006 ◽

Vol 86 (4) ◽

pp. 1093-1131 ◽

Cited By ~ 233

Author(s):

Antonio G. García ◽

Antonio M. García-De-Diego ◽

Luis Gandía ◽

Ricardo Borges ◽

Javier García-Sancho

Keyword(s):

Endoplasmic Reticulum ◽

Calcium Channels ◽

Calcium Channel ◽

Chromaffin Cells ◽

Active Sites ◽

Cultured Cells ◽

Mammalian Species ◽

Opiate Receptors ◽

Catecholamine Release ◽

High K

At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration ([Ca2+]c) depends on at least four efficient regulatory systems: 1) plasmalemmal calcium channels, 2) endoplasmic reticulum, 3) mitochondria, and 4) chromaffin vesicles. Different mammalian species express different levels of the L, N, P/Q, and R subtypes of high-voltage-activated calcium channels; in bovine and humans, P/Q channels predominate, whereas in felines and murine species, L-type channels predominate. The calcium channels in chromaffin cells are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Chromaffin cells have been particularly useful in studying calcium channel current autoregulation by materials coreleased with catecholamines, such as ATP and opiates. Depending on the preparation (cultured cells, adrenal slices) and the stimulation pattern (action potentials, depolarizing pulses, high K+, acetylcholine), the role of each calcium channel in controlling catecholamine release can change drastically. Targeted aequorin and confocal microscopy shows that Ca2+ entry through calcium channels can refill the endoplasmic reticulum (ER) to nearly millimolar concentrations, and causes the release of Ca2+ (CICR). Depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates catecholamine release. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients, upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 to ∼50 μM, triggering CICR, mitochondrial Ca2+ uptake, and exocytosis at nearby secretory active sites. The fact that protonophores abolish mitochondrial Ca2+ uptake, and increase catecholamine release three- to fivefold, support the earlier observation. This increase is probably due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; this transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR, and/or mitochondrial Ca2+ release. We propose that chromaffin cells have developed functional triads that are formed by calcium channels, the ER, and the mitochondria and locally control the [Ca2+]c that regulate the early and late steps of exocytosis.

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