Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones

doi:10.1016/S0143-4160(02)00094-5

Cell Calcium

Volume 32, Issue 1, July 2002, Pages 49-52

https://doi.org/10.1016/S0143-4160(02)00094-5 Get rights and content

Abstract

The action of Xestospongin C (XeC) on calcium concentration in the cytosol ([Ca²⁺]_i) and within the lumen of endoplasmic reticulum (ER) ([Ca²⁺]_L) was studied using cultured dorsal root ganglia (DRG) neurones. Application of 2.5 μM of XeC triggered a slow [Ca²⁺]_i transient as measured by Fura-2 video-imaging. The kinetics and amplitude of XeC-induced [Ca²⁺]_i response was similar to that triggered by 1 μM thapsigargin (TG). The [Ca²⁺]_L was monitored in cells loaded with low-affinity Ca²⁺ indicator Mag-Fura-2. The cytosolic portion of Mag-Fura-2 was removed by permeabilisation of the plasmalemma with saponin. Application of XeC to these permeabilised neurones resulted in a slow depletion of the ER Ca²⁺ store. XeC, however, failed to inhibit inositol 1,4,5-trisphosphate (InsP₃)-induced [Ca²⁺]_L responses. We conclude that XeC is a potent inhibitor of sarco(endo)plasmic reticulum calcium ATPase, and it cannot be regarded as a specific inhibitor of InsP₃ receptors in cultured DRG neurones.

Section snippets

INTRODUCTION

Endoplasmic reticulum (ER) calcium stores are an indispensable source for calcium signalling in most eukaryotic cells 1., 2., 3., 4.. Mobilisation of Ca²⁺ from these stores results from the activation of two families of Ca²⁺ release channels, ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (InsP₃Rs; for review see 3., 5., 6., 7.). Under physiological conditions, RyRs are activated by cytosolic calcium, thus producing Ca²⁺-induced Ca²⁺ release, whereas InsP₃Rs are

MATERIALS AND METHODS

DRG neurones were enzymatically isolated from new-born (1–3 days old) Sprague–Dawley rats using a conventional treatment with 0.1% protease (type XIV) in HEPES-buffered Minimum Essential Eagle Medium (MEM) for 8 min at 37 °C. Individual cells were separated mechanically and plated on poly-L-ornithine (1 mg/ml) and laminin (0.01 mg/ml) covered glass coverslips. Neurones were maintained in culture media (DMEM, supplemented with 10% horse serum, 50 U/ml penicillin/streptomycin mixture and 6 μg/ml

RESULTS AND DISCUSSION

We began by investigating the action of XeC on [Ca²⁺]_i in isolated DRG neurones. Extracellular application of 2.5 μM XeC dissolved in normal physiological solution caused slow elevation of [Ca²⁺]_i in all cells tested (n=9, Fig. 1A). XeC elevated [Ca²⁺]_i by 136±31 nM. After reaching the peak level (which was attained 100–150 s after the beginning of XeC application) [Ca²⁺]_i declined slowly to the resting level. Very similar [Ca²⁺]_i dynamics were observed when cells were challenged with TG, an

Acknowledgements

This research was supported by a BBSRC research grant to AV (ref. 34/C12751).

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^☆: Dr A. Parekh (Oxford University) acted as an invited editor for this paper.

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Short communicationXestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones☆

Abstract

Section snippets

INTRODUCTION

MATERIALS AND METHODS

RESULTS AND DISCUSSION

Acknowledgements

Prog. Neurobiol.

Cell Calcium

Trends Neurosci.

Cell Calcium

Neuron

Cell Calcium

Trends Neurosci.

Cell

Neuron

J. Biol. Chem.

Mol. Cell. Endocrinol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Short communication
Xestospongin C empties the ER calcium store but does not inhibit InsP₃-induced Ca²⁺ release in cultured dorsal root ganglia neurones☆