Xestospongin C empties the ER calcium store but does not inhibit InsP3-induced Ca2+ release in cultured dorsal root ganglia neurones.
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 microM 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 microM 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.
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ABSTRACT: Background and PurposeInositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels. Interactions of the commonly used antagonists of IP3Rs with IP3R subtypes are poorly understood.Experimental ApproachIP3-evoked Ca2+ release from permeabilized DT40 cells stably expressing single subtypes of mammalian IP3R was measured using a luminal Ca2+ indicator. The effects of commonly used antagonists on IP3-evoked Ca2+ release and 3H-IP3 binding were characterized.Key ResultsFunctional analyses showed that heparin was a competitive antagonist of all IP3R subtypes with different affinities for each (IP3R3>IP3R1≥IP3R2). This sequence did not match the affinities for heparin binding to the isolated N-terminal from each IP3R subtype. 2-aminoethoxydiphenyl borate (2-APB) and high concentrations of caffeine selectively inhibited IP3R1 without affecting IP3 binding. Neither Xestospongin C nor Xestospongin D effectively inhibited IP3-evoked Ca2+ release via any IP3R subtype.Conclusions and ImplicationsHeparin competes with IP3, but its access to the IP3-binding core is substantially hindered by additional IP3R residues. These interactions may contribute to its modest selectivity for IP3R3. Practicable concentrations of caffeine and 2-APB inhibit only IP3R1. Xestospongins do not appear to be effective antagonists of IP3Rs.British Journal of Pharmacology 03/2014; · 5.07 Impact Factor
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ABSTRACT: Persistent inflammation results in an increase in the magnitude and duration of high K(+)-evoked Ca(2+) transients in putative nociceptive cutaneous dorsal root ganglion (DRG) neurons. The purpose of the present study was to determine whether recruitment of Ca(2+)-induced Ca(2+) release (CICR) contributes to these inflammation-induced changes. Acutely dissociated, retrogradely labeled cutaneous DRG neurons from naïve and complete Freund's adjuvant inflamed adult male Sprague-Dawley rats were studied with ratiometric microfluorimetry. Ryanodine only attenuated the duration but not magnitude of the high K(+)-evoked Ca(2+) transient in neurons from inflamed rats. However, there was no significant impact of inflammation on the potency or efficacy of ryanodine-induced block of the caffeine-evoked Ca(2+) transient, or the impact of sarco-endoplasmic reticulum ATPase (SERCA) inhibition on the high K(+)-evoked Ca(2+) transient. Furthermore, while there was no change in the magnitude, an inflammation-induced increase in the duration of the caffeine-evoked Ca(2+) transient was only observed with a prolonged caffeine application. In contrast to the high K(+)-evoked Ca(2+) transient, there was no evidence of direct mitochondrial involvement or that of the Ca(2+) extrusion mechanism, the Na(+)/Ca(2+) exchanger, on the caffeine-evoked Ca(2+) transient, and block of SERCA only increased the duration of this transient. These results indicate the presence of Ca(2+) regulatory domains in cutaneous nociceptive DRG neurons within which cytosolic Ca(2+) increased via influx and release are highly segregated. Furthermore, our results suggest that changes in neither CICR machinery nor the coupling between Ca(2+) influx and CICR are primarily responsible for the inflammation-induced changes in the evoked Ca(2+) transient.Cell calcium 05/2013; · 4.29 Impact Factor
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ABSTRACT: 1,4,5-Inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) mediate release of Ca(2+) from internal stores in many neurons. The details of the spatial and temporal characteristics of these signals and their interactions in dendrites remain to be clarified. We found that localized Ca(2+) release events, with no associated change in membrane potential, occurred spontaneously in the dendrites of rat hippocampal CA1 pyramidal neurons. Their rate, but not their amplitude or time course, could be modulated by changes in membrane potential. Together, these results suggest that the spontaneous events are similar to RyR-dependent Ca(2+) "sparks" found in cardiac myocytes. In addition, we found that we could generate another kind of localized Ca(2+) release event by either a synaptic tetanus in the presence of 3-((R)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid and CNQX or by uncaging IP3. These events had slower rise times and decay times than sparks and were more heterogeneous. These properties are similar to Ca(2+) "puffs" found in oocytes. These two localized signals interact. Low-intensity tetanic synaptic stimulation or uncaging of IP3 increased the decay time of spontaneous Ca(2+) events without changing their rise time or amplitude. Pharmacological experiments suggest that this event widening is attributable to a delayed IP3R-mediated release of Ca(2+) triggered by the synergistic action of IP3 and Ca(2+) released by RyRs. The actions of IP3 appear to be confined to the main apical dendrite because uncaging IP3 in the oblique dendrites has no effect on the time course of localized events or backpropagating action potential-evoked Ca(2+) signals in this region.Journal of Neuroscience 11/2013; 33(45):17777-88. · 6.75 Impact Factor
Cell Calcium (2002) 32(1), 49–52
0143-4160/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0143-4160(02)00094-5, available online at http://www.idealibrary.com on
Xestospongin C empties the ER
calcium store but does not inhibit
InsP3-induced Ca2+release in cultured
dorsal root ganglia neurones?
N. Solovyova,1P. Fernyhough,1G. Glazner,2A. Verkhratsky1
1School of Biological Sciences, The University of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13 9PT, UK
2University of Manitoba and St. Boniface Hospital Research Centre R4052-351, Tache Avenue, Winnipeg, MB, Canada R2H 2A6
Summary 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?M of XeC triggered a slow [Ca2+]itransient as measured by Fura-2 video-imaging. The kinetics and amplitude of
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+]Lresponses. 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 InsP3receptors in cultured DRG neurones.
© 2002 Elsevier Science Ltd. All rights reserved.
Endoplasmic reticulum (ER) calcium stores are an indis-
pensable source for calcium signalling in most eukary-
otic cells [1–4]. Mobilisation of Ca2+from these stores
results from the activation of two families of Ca2+re-
lease channels, ryanodine receptors (RyRs) and inositol
1,4,5-trisphosphate receptors (InsP3Rs; for review see
[3,5–7]). Under physiological conditions, RyRs are acti-
vated by cytosolic calcium, thus producing Ca2+-induced
Ca2+release, whereas InsP3Rs are stimulated by InsP3
generated by phospholipase C, which in turn is controlled
by numerous metabotropic receptors.
Although the availability of specific drugs which inter-
act with these receptors is important, the pharmacology
of Ca2+release channels remains confusing. For exam-
ple, heparin, widely used as an InsP3R inhibitor, activates
Received 1 March 2002
Accepted 24 April 2002
Correspondence to: Dr A. Verkhratsky, School of Biological Sciences, The Uni-
versity of Manchester, 1.124 Stopford Building, Oxford Road, Manchester M13
9PT, UK. Tel.: +44-161-2755414; fax: +44-161-275-5948;
?Dr A. Parekh (Oxford University) acted as an invited editor for this paper.
RyRs , whereas another potential blocker of InsP3Rs,
sarco(endo)plasmic reticulum calcium ATPase (SERCA)
. Moreover, caffeine, generally considered an activator
of RyR , also inhibits InsP3 in millimolar concentra-
tions [11,12]. Promising new compounds which could
selectively inhibit InsP3R appeared with the discovery of
xestospongins (A–D), which have been shown to inhibit
InsP3-mediated Ca2+release with very high potency (IC50
ranging between 358nM to 5.9?M; ). This stimulated
usage of the most potent member of this family, Xestosop-
ngin C (XeC), as a selective InsP3R inhibitor in various tis-
sues and experimental protocols (e.g. [14–25]). Yet, direct
physiological data demonstrating the action of XeC on
InsP3-mediated Ca2+release are scarce, being essentially
limited to the initial publication in Neuron .
In 1999 Cell Calcium published a manuscript by De
Smet et al. , which questioned the specificity of XeC
by demonstrating that this drug interacts with SERCA
pumps. This observation was further substantiated by
Castonguay and Robitaille , who presented physi-
ological data demonstrating that XeC inhibits SERCA
pumps at the neuromuscular junction and Schwann cells.
The question highlighted by these two papers is very
N Solovyova, P Fernyhough, G Glazner, A Verkhratsky
important, as it undermines the usage of XeC as a selec-
tive blocker of InsP3Rs. We, therefore, decided to address
this problem directly by investigating how XeC acts on
cytosolic free calcium ([Ca2+]i) and how it affects free
Ca2+concentration within the ER lumen ([Ca2+]L) in an-
other preparation, cultured DRG neurones. We found that
XeC depletes ER Ca2+store in a manner similar to thap-
sigargin (TG), but does not affect InsP3-activated Ca2+
MATERIALS AND METHODS
DRG neurones were enzymatically isolated from new-born
(1–3 days old) Sprague–Dawley rats using a conven-
tional treatment with 0.1% protease (type XIV) in HEPES-
buffered Minimum Essential Eagle Medium (MEM) for
8min at 37◦C. Individual cells were separated mechani-
cally and plated on poly-L-ornithine (1mg/ml) and laminin
(0.01mg/ml) covered glass coverslips. Neurones were
maintained in culture media (DMEM, supplemented with
10% horse serum, 50U/ml penicillin/streptomycin mix-
ture and 6?g/ml insulin) at 37◦C in 95% air +5% CO2for
1–2 days prior to experimental treatment.
Fig. 1 XeC action on [Ca2+]iand [Ca2+]Lin cultured DRG neurones. (A) Example of [Ca2+]irecording from a Fura-2/AM loaded DRG
neurone treated with ATP (10?M, 10-s application, shown by an arrow) and XeC (2.5?M). (B) The [Ca2+]itrace taken from another neurone
similarly challenged with ATP and TG (1?M). Note the similarity between XeC- and TG-induced [Ca2+]iresponses. (C) Intra-ER free Ca2+
dynamics in response to InsP3and XeC applications in a permeabilised DRG neurone pre-loaded with Mag-Fura-2. The drugs were applied
as shown in the graph. Note that InsP3triggers Ca2+release in the presence of XeC, however, [Ca2+]Ldoes not recover after InsP3removal.
The cytosolic calcium concentration, [Ca2+]I, was mea-
sured using a conventional Fura-2 technique (see ).
[Ca2+]L, cells were loaded with low-affinity Ca2+probe,
Mag-Fura-2, by incubation with the membrane permeable
form of the dye (5?M Mag-Fura-2/AM for 20min at 37◦C,
followed by 60min washout with normal physiological so-
lution at 37◦C), so that the probe was trapped within both
intracellular organelles and the cytoplasm. Normal phys-
iological solution comprised (in mM): NaCl 135, KCl 3,
CaCl22, MgCl21, HEPES/NaOH 20, glucose 20; pH 7.4. To
remove the cytoplasmic portion of the dye and gain direct
access to intracellular Ca2+release channels, the cellular
membrane was permeabilised by brief (7–10s) application
of saponin (0.001%) in ‘intracellular’ solution (in mM:
KCl 135, ATP 3, MgCl22, CaCl20.4, EGTA 1, HEPES/KOH
20; pH 7.2; free Ca2+concentration 100nM, free ATP con-
centration 1mM and free Mg2+concentration 0.1mM).
Fluorescence images were captured using an Olympus
IX70 inverted epifluorescence microscope (40× UV ob-
jective) equipped with a charge-coupled device, cooled
intensified camera (Pentamax Gene IV, Roper Scientific,
UK). The specimen was alternately illuminated at 340 and
Cell Calcium (2002) 32(1), 49–52© 2002 Elsevier Science Ltd. All rights reserved.
Xestospongin C is a potent inhibitor of ER calcium but not of InsP3receptors in DRG neurones
380nm by a monochromator (Polychrom IV, TILL Pho-
tonics, Germany) at a cycle frequency of 1–5Hz. Control
over the experiment, image storage and off-line analysis
was performed by the use of MetaFluor/MetaMorph soft-
ware (Universal Imaging Corporation, USA) running on a
Windows 98 workstation.
The [Ca2+]Lvalues were calculated using the 340/380
ratio with the equation [Ca2+]L= K∗(R − Rmin)/(Rmax− R).
The Rmin, Rmax and K*were determined using exposure
of permeabilised neurones to 20?M ionomycin and three
calibrating solutions with [Ca2+] < 10nM (10mM EGTA),
100?M and 10mM. Values of Rmin, Rmaxand K*were 0.35,
1.7 and 276?M, respectively.
All solutions were applied using a fast local superfusion
technique , which ensured complete exchange of the
milieu surrounding the cell within 100ms. All reagents
were purchased from Sigma (Dorset, UK), and fluorescent
probes were obtained from Molecular Probes (Oregon,
USA). All data are presented as mean ± S.D.
RESULTS AND DISCUSSION
We began by investigating the action of XeC on [Ca2+]i
in isolated DRG neurones. Extracellular application of
2.5?M XeC dissolved in normal physiological solution
caused slow elevation of [Ca2+]iin all cells tested (n = 9,
Fig. 1A). XeC elevated [Ca2+]i by 136 ± 31nM. After
reaching the peak level (which was attained 100–150s
after the beginning of XeC application) [Ca2+]i declined
slowly to the resting level. Very similar [Ca2+]i dynam-
ics were observed when cells were challenged with TG,
an inhibitor of SERCA pumps. Application of 1?M TG
triggered a slow increase in [Ca2+]i, with an average am-
plitude of 122 ± 41nM (n = 8). This TG-induced [Ca2+]i
transient peaked within ∼70–100s after administra-
tion of the drug, afterwards [Ca2+]i slowly recovered
towards the resting values (Fig. 1B). The striking simi-
larity in the action of XeC and TG on [Ca2+]i led us to
hypothesise that XeC inhibits SERCA pumps, thus re-
leasing Ca2+from the ER lumen due to an unopposed
We tested this possibility by monitoring intra-ER free
Ca2+dynamics in response to applications of InsP3 and
XeC to permeabilised DRG neurones, in which the ER
contained the low-affinity Ca2+indicator Mag-Fura-2.
Application of InsP3at a supramaximal (10?M) concen-
tration triggered a rapid decrease in [Ca2+]L(Fig. 1C). After
removal of InsP3, [Ca2+]Lrecovered to the pre-stimulated
value due to SERCA-dependent uptake. Application of
2.5?M XeC resulted in a slow decline in [Ca2+]L, which
decreased from 321 ± 74 to 205 ± 66?M (n = 7) within
300s. Subsequent application of 10?M InsP3, however,
triggered a rapid decline in [Ca2+]L to approximately
120?M, where it stabilised. Upon removal of InsP3,
[Ca2+]L showed no signs of recovery in all cells tested
(n = 7).
Our observations support findings by De Smet et al. 
and by Castonguay and Robitaille  pointing to the ab-
sence of specificity of XeC. The action of XeC on cytosolic
calcium concentration in DRG neurones is very similar to
the action of TG. Both compounds trigger a slow transient
[Ca2+]irise, which is compatible with ER depletion follow-
ing inhibition of SERCA pumps. Furthermore, we found
that XeC induced a slow depletion of intra-ER Ca2+in a
manner similar to TG (see ). Moreover, in the same ex-
periments, we failed to find an inhibitory action of XeC on
Initial data on XeC action on Ca2+signals in PC12
cells and in cultured astrocytes, which were interpreted
as XeC-dependent inhibition of InsP3Rs , can be ex-
plained in terms of SERCA inhibition. Indeed, in those
initial experiments, XeC-dependent inhibition of brady-
long-lasting (10min) treatment with a very high (20?M)
concentration of the drug. Such a treatment with XeC
could effectively deplete the ER store, thus resulting in
inhibition of Ca2+response. In conclusion, our results
suggest that XeC is a potent inhibitor of SERCA pumps in
DRG neurones and is not a specific antagonist of InsP3Rs.
This research was supported by a BBSRC research grant to
AV (ref. 34/C12751).
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