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Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells

  • Signaling and cell physiology
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Abstract

Gasotransmitter hydrogen sulphide (H2S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H2S) and GYY4137 (GYY, slow donor of H2S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K+-depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca2+ entry through voltage-activated Ca2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca2+]c and the K+-elicited [Ca2+]c transients; such effects of H2S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K+ in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca2+ handling by the ER and mitochondria. On the basis of these results, we propose that H2S regulates catecholamine secretory responses triggered by K+ in BCCs by (i) mobilisation of ER Ca2+ and (ii) interference with mitochondrial Ca2+ circulation. In so doing, the clearance of the [Ca2+]c transient will be delayed and the Ca2+-dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis.

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Abbreviations

H2S:

Hydrogen sulphide

GYY:

GYY4137; morpholin-4-ium 4-methoxyphenyl(morpholino) phosphinodithioate; a slow releasing H2S donor

NaHS:

Sodium hydrosulphide; a fast releasing H2S donor

VACCs:

Voltage-activated calcium channels

DRG:

Dorsal root ganglion

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Funding

Supported by grants from MINECO, Spain (SAF 2013-44108-P and SAF 2016-78892-R). Also by CABYCIC UAM/Bioibérica and Nutrinfant UAM/Alter, Spain. The authors also thank the continued support of Fundación Teófilo Hernando, Madrid, Spain.

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Authors and Affiliations

  1. Instituto Teófilo Hernando, Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Arzobispo Morcillo, 4, 28029, Madrid, Spain

    Ricardo de Pascual, Andrés M. Baraibar, Iago Méndez-López, Martín Pérez-Ciria, Ignacio Polo-Vaquero, Luis Gandía, Antonio G. García & Antonio M. G. de Diego

  2. Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Madrid, Spain

    Iago Méndez-López, Antonio G. García & Antonio M. G. de Diego

  3. Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Texas Southern University, 3100 Cleburne Street, Houston, TX, 77004, USA

    Sunny E. Ohia

  4. DNS Neuroscience, Parque Científico de Madrid, Madrid, Spain

    Antonio G. García & Antonio M. G. de Diego

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  1. Ricardo de Pascual
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  2. Andrés M. Baraibar
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Correspondence to Antonio G. García.

Electronic supplementary material

Supplementary Fig. 1

Compounds NaHS and GYY do not affect inward sodium currents (INa). a, c Original INa traces obtained in BCCs that where voltage-clamped at − 80 mV, following the protocol on the top (test depolarizing pulses of 10-ms duration applied at 10-s intervals). Traces were obtained before (control), after 2 min in the presence of 100 μM each of NaHS or GYY, and 3 min after washout of the compounds (WO). b, d Averaged pooled data (mean ± SEM) of the number of experiments shown in parentheses (number of cells and cultures). (JPG 430 kb)

Supplementary Fig. 2

Mild cell depolarization of BCCs produced by NaHS, but not by GYY. Membrane potential (Vm) was recorded under the current-clamp configuration of the patch-clamp technique. a, d Vm recording from two example cells exposed to NaHS (a) or GYY (d) during the time period indicated by the horizontal bar. b, c, e, f Pooled data in control conditions, after 2-min exposure to 100 or 300 μM NaHS (b, c) or GYY (e, f), and after compounds washout. Data are means ± SEM of the number of cells and cultures given in parentheses. *p < 0.05, *** p < 0.001 with respect to control currents (JPG 403 kb)

Supplementary Fig. 3

Evoked action potentials (APs) are modified by NaHS, but not by GYY. Single APs were evoked by threshold depolarisations in current-clamped BCCs. a, d AP traces obtained in example cells before (control) and 1 min after cell exposure to NaHS (a) or GYY (d). b, c, e, f Pooled data on the normalised AP peak amplitude and after-hyperpolarisation (AHP). Data are means ± SEM of the number of cells and cultures shown in parentheses. *p < 0.05, ***p < 0.001, compared with control (JPG 617 kb)

Supplementary Fig. 4

Monitoring of the cytosolic Ca2+ concentrations ([Ca2+]c) in BCC populations plated in 96-well black plates and loaded with fura-2-AM. The use in these experiments of fura-2 allowed the conversion of arbitrary fluorescence units into nM [Ca2+] (ordinates). Averaged basal [Ca2+]c varied from around 75 to 120 nM. In order to compare traces (a), the initial basal fluorescence has been subtracted. Therefore, values in this panel are actually Δ[Ca2+]c (ordinates). NaHS at the final concentrations (μM) shown in each curve elicited a gradual increase of [Ca2+]c that reached a similar plateau at the three concentrations tested. b Time course of the variation of [Ca2+]c in cells exposed first to NaHS (10 μM) and after 3 min, to cyclopiazonic acid (CPA, 10 μM) added on top of NaHS. c [Ca2+]c elevations produced by adding first CPA (10 μM); 3 min later, NaHS (10 μM) was added on top of CPA. Data shown in all panels are averages of 3–5 experiments. (JPG 444 kb)

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de Pascual, R., Baraibar, A.M., Méndez-López, I. et al. Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells. Pflugers Arch - Eur J Physiol 470, 1255–1270 (2018). https://doi.org/10.1007/s00424-018-2147-7

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