Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes

doi:10.1016/S0143-4160(96)90094-9

Cell Calcium

Volume 20, Issue 6, December 1996, Pages 515-524

https://doi.org/10.1016/S0143-4160(96)90094-9 Get rights and content

Abstract

Intracellular Ca²⁺ ([Ca²⁺]_i) dynamics were studied in identified rat gonadotropes using the whole-cell patch-clamp technique in conjunction with Indo-1 photometry. The kinetics of depolarization-induced [Ca²⁺]_i transients vary with Ca²⁺ load. In addition to a rapid initial decay, large (> 500 nM) [Ca²⁺]_i transients have a slow plateau phase. Application of the mitochondrial inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP) significantly slows the decay of [Ca²⁺]_i transients, consistent with stopping uptake of Ca²⁺ by mitochondria. CCCP causes a small increase of [Ca²⁺]_i at rest. After a large Ca²⁺ entry the amount is much larger, consistent with release from a mitochondrial Ca²⁺ pool that fills during cytoplasmic Ca²⁺ loading. The rate of Ca²⁺ uptake by mitochondria is dependent upon [Ca ²⁺]_i. Consistent with previous studies, gonadotropin releasing hormone (GnRH) induces [Ca²⁺]_i oscillations. The mitochondrial inhibitors CCCP and cyanide (CN⁻) terminate these oscillations. The mitochondrial ATP-synthase inhibitor oligomycin reduces the frequency and increases the amplitude of the oscillations. In the presence of ruthenium red (a non-specific blocker of the mitochondrial Ca²⁺-uniporter) in the pipette, GnRH does not induce rhythmic [Ca²⁺]_i oscillations. We suggest that mitochondria play a significant role in the rapid clearance of cytosolic Ca²⁺ loads in gonadotropes and participate in GnRH-induced periodic [Ca²⁺]_i oscillations.

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In the cochlea, cell damage triggers intercellular Ca²⁺ waves that propagate through the glial-like supporting cells that surround receptor hair cells. These Ca²⁺ waves are thought to convey information about sensory hair cell-damage to the surrounding supporting cells within the cochlear epithelium. Mitochondria are key regulators of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt), and yet little is known about their role during the propagation of such intercellular Ca²⁺ signalling. Using neonatal rat cochlear explants and fluorescence imaging techniques, we explore how mitochondria modulate supporting cell [Ca²⁺]_cyt signals that are triggered by ATP or by hair cell damage. ATP application (0.1–50 μM) caused a dose dependent increase in [Ca²⁺]_cyt which was accompanied by an increase in mitochondrial calcium. Blocking mitochondrial Ca²⁺ uptake by dissipating the mitochondrial membrane potential using CCCP and oligomycin or using Ru360, an inhibitor of the mitochondrial Ca²⁺ uniporter, enhanced the peak amplitude and duration of ATP-induced [Ca²⁺]_cyt transients. In the presence of Ru360, the mean propagation velocity, amplitude and extent of spread of damage-induced intercellular Ca²⁺ waves was significantly increased. Thus, mitochondria function as spatial Ca²⁺ buffers during agonist-evoked [Ca²⁺]_cyt signalling in cochlear supporting cells and play a significant role in regulating the spatio-temporal properties of intercellular Ca²⁺ waves.
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Airway myocytes are the primary effectors of airway reactivity which modulates airway resistance and hence ventilation. Stimulation of airway myocytes results in an increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and the subsequent activation of the contractile apparatus. Many contractile agonists, including acetylcholine, induce [Ca²⁺]_i increase via Ca²⁺ release from the sarcoplasmic reticulum through InsP₃ receptors. Several models have been developed to explain the characteristics of InsP₃-induced [Ca²⁺]_i responses, in particular Ca²⁺ oscillations. The article reviews the modelling of the major structures implicated in intracellular Ca²⁺ handling, i.e., InsP₃ receptors, SERCAs, mitochondria and Ca²⁺-binding cytosolic proteins. We developed theoretical models specifically dedicated to the airway myocyte which include the major mechanisms responsible for intracellular Ca²⁺ handling identified in these cells. These biocomputations pointed out the importance of the relative proportion of InsP₃ receptor isoforms and the respective role of the different mechanisms responsible for cytosolic Ca²⁺ clearance in the pattern of [Ca²⁺]_i variations. We have developed a theoretical model of membrane conductances that predicts the variations in membrane potential and extracellular Ca²⁺ influx. Stimulation of this model by simulated increase in [Ca²⁺]_i predicts membrane depolarisation, but not great enough to trigger a significant opening of voltage-dependant Ca²⁺ channels. This may explain why airway contraction induced by cholinergic stimulation does not greatly depend on extracellular calcium. The development of such models of airway myocytes is important for the understanding of the cellular mechanisms of airway reactivity and their possible modulation by pharmacological agents.
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Goldfish somatotropes contain multiple functionally distinct classes of non-mitochondrial intracellular Ca²⁺ stores. In this study, we investigated the role of mitochondrial Ca²⁺ handling in the control of hormone secretion. Inhibition of mitochondrial Ca²⁺ uptake with 10 μM ruthenium red (RR) and 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP) caused a small and reversible increase in cytosolic [Ca²⁺]. Despite relatively modest global Ca²⁺ signals, RR and CCCP stimulated robust GH secretion under basal culture conditions. CCCP-stimulated hormone release was abolished in cells pre-incubated with 50 μM BAPTA-AM, suggesting that elevations in cytosolic [Ca²⁺] mediate this release of GH. Both caffeine-sensitive intracellular Ca²⁺ stores and l-type Ca²⁺ channels can be the source of the Ca²⁺ buffered by mitochondria in somatotropes. The stimulatory effect of RR on caffeine-stimulated GH release was enhanced dramatically in the presence of ryanodine, pointing to a complex interaction between these three Ca²⁺ stores. Inhibition of mitochondrial Ca²⁺ uptake with RR augmented GH release evoked by only one of the two endogenous gonadotropin-releasing hormones. Thus, we provide the first evidence that mitochondrial Ca²⁺ buffering is differentially involved in specific agonist Ca²⁺ signaling pathways and plays an important role in the control of basal GH release.

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ResearchInvolvement of mitochondria in intracellular calcium sequestration by rat gonadotropes

Abstract

Curr Top Cell Regul

Prog Neurobiol

J Biol Chem

J Biol Chem

Neuron

J Biol Chem

Neuron

Pharmacol Ther

Brain Res

Cell

GnRH-induced Ca2+ oscillations and rhythmic hyperpolarizations of pituitary gonadotropes

Science

Integration of cytoplasmic calcium and membrane potential oscillations maintain calcium signaling in pituitary gonadotrophs

Rhythmic exocytosis stimulated by GnRH-induced calcium oscillations in rat gonadotropes

Science

Minimal model for signal-induced Ca2+ oscillations and for their frequency encoding through protein phosphorylation

Calcium oscillations in pituitary gonadotrophs: comparison of experiment and theory

Calcium homeostasis in identified rat gonadotropes

J Physiol Lond

Cyclic Call changes in intracellular stores of gonadotropes during gonadotropen-releasing hormone-stimulated Call oscillations

Mechanisms by which mitochondria transport calcium

Am J Physiol

Research
Involvement of mitochondria in intracellular calcium sequestration by rat gonadotropes

GnRH-induced Ca²⁺ oscillations and rhythmic hyperpolarizations of pituitary gonadotropes

Minimal model for signal-induced Ca²⁺ oscillations and for their frequency encoding through protein phosphorylation