G\xce\xb1i/o-dependent Ca2+\nmobilization and G\xce\xb1q-dependent PKC\xce\xb1 regulation of\nCa2+-sensing receptor-mediated responses in N18TG2\nneuroblastoma cells
A functional Ca2+-sensing receptor (CaS) is expressed endogenously in mouse N18TG2 neuroblastoma cells, and sequence analysis of the cDNA indicates high homology with both rat and human parathyroid CaS cDNAs. The CaS in N18TG2 cells appears as a single immunoreactive protein band at about 150 kDa on Western blots, consistent with native CaS from dorsal root ganglia. Both wild type (WT) and G\xce\xb1q antisense knock-down (KD) cells responded to Ca2+ and calindol, a positive allosteric modulator of the CaS, with a transient increase in intracellular Ca2+ concentration ([Ca2+]i), which was larger in the G\xce\xb1q KD cells. Stimulation with 1 mM extracellular Ca2+ (Ca2+e) increased [Ca2+]i in N18TG2 G\xce\xb1q KD compared to WT cells. Ca2+ mobilization was dependent on pertussis toxin-sensitive G\xce\xb1i/o proteins and reduced by 30 \xce\xbcM 2-amino-ethyldiphenyl borate and 50 \xce\xbcM nifedipine to the same plateau levels in both cell types. Membrane-associated PKC\xce\xb1 and p-PKC\xce\xb1 increased with increasing [Ca2+]e in WT cells, but decreased in G\xce\xb1q KD cells. Treatment of cells with 1 \xce\xbcM G\xd3\xa7 6976, a Ca2+-specific PKC inhibitor reduced Ca2+ mobilization and membrane-associated PKC\xce\xb1 and p-PKC\xce\xb1 in both cell types. The results indicate that the CaS-mediated increase in [Ca2+]i in N18TG2 cells is dependent on G\xce\xb1i/o proteins via inositol-1,4,5- triphosphate (IP3) channels and store-operated Ca2+ entry channels, whereas modulation of CaS responses involving PKC\xce\xb1 phosphorylation and translocation to the plasma membrane occurs via a G\xce\xb1q mechanism.
Introduction<!>Materials<!>Cell culture<!>Expression Analysis of CaS and PKC isoforms in N18TG2 Cells<!>Intracellular Ca2+ measurements<!>Ca2+-induced membrane translocation and phosphorylation of\nPKC\xce\xb1<!>Statistical analysis<!>Expression of a neuronal CaS in N18TG2 cells<!>Microfluorimetric analysis of changes in\n[Ca2+]i in N18TG2 cells\nfollowing stimulation with [Ca2+]e\nor calindol, a positive allosteric modulator of CaS<!>Pertussis toxin (PTX)-sensitive G\xce\xb1i/o proteins are\nrequired for CaS-mediated mobilization of Ca2+ in N18TG2\ncells<!>Ion channels involved in CaS-mediated changes in\n[Ca2+]i in N18TG2\ncells<!>Regulation of CaS-mediated changes in\n[Ca2+]i by PKC in N18TG2\ncells<!>Discussion
<p>Since the initial cloning of the Ca2+ -sensing receptor
(CaS; official IUPHAR name) from bovine parathyroid gland (Brown 1999, Brown
et al. 1993, Brown
& MacLeod 2001)), nervous tissue from rat has been found to
express a full-length, alternatively spliced form of the receptor, which is
concentrated in nerve terminals and involved in the regulation of neuronal cell
growth and migration during development, synaptic plasticity and neurotransmission
in mature nerve terminals [for review, see (Bouschet & Henley 2005, Bouschet
et al. 2005), (Ruat
& Traiffort 2013)]. In addition to the brain (Ruat et al. 1995), the CaS is
also expressed in perivascular sensory nerves (Bukoski 1998, Bukoski et
al. 1997, Wang &
Bukoski 1998, Wang & Bukoski
1999), trigeminal ganglia and sensory axons (Heyeraas et al. 2008). We reported the
cloning and sequencing of the dorsal root ganglion (DRG) CaS message and found
significant homology with the rat kidney CaS cDNA (Wang et al. 2003b). Expression analysis of a DRG
CaS-EGFP fusion protein transfected into HEK293 cells showed that the fusion protein
incorporates into the cell membranes and is functionally linked to a transient
increase in [Ca2+]i (Awumey et al. 2007). Activation of the
CaS expressed in DRG and perivascular sensory nerves (Bukoski et al. 1997, Ishioka & Bukoski 1999) by extracellular Ca2+
(Ca2+e) results in the release of a vasodilator
transmitter, possibly an endocannabinoid (Awumey
et al. 2008, Bukoski
1998) (Bukoski et al.
2002, Ishioka & Bukoski
1999).</p><p>As a G protein-coupled receptor (GPCR), the CaS can couple to more than one
type of GÎ± subunit and influence the properties of GÎ²Î³
signaling (Neves et al.
2002). Three modes of CaS coupling to G proteins have been reported, namely
through: i) GÎ±i to inhibit adenylyl cyclase (AC) and activate
mitogen activated protein kinase (MAPK); ii) GÎ±q to stimulate
phospholipase C (PLC) and phospholipase A2 (PLA2); and iii)
GÎ²Î³ to stimulate phosphoinositide-3-kinase (Brown & MacLeod 2001). Activation of the CaS by
Ca2+e, other polyvalent cations or allosteric
regulators stimulates PLC, PLD, or PLA signaling pathways depending on the cell type
(for review, see (Conigrave & Ward
2013
Breitwieser 2013). PLC activation results in
the generation of inositol-1,4,5-trisphosphate (IP3) and the release of
Ca2+ from the endoplasmic reticulum (ER). It has been
difficult to assess the roles of Gi/o versus Gq in activation
of the neuronal CaS because there have been no established neuronal cell models for
studying receptor coupling to intracellular signal transduction events. N18TG2
cells, a mouse neuroblastoma clone, express many properties of neurons (Mukhopadhyay et al. 2002), and
have been shown to produce the endocannabinoid, 2-arachidonoylglycerol (2-AG) in
response to elevations in [Ca2+]i
(Bisogno et al. 1997).
Using this established neuronal cell model, the present study describes the
signaling mechanisms of the endogenously-expressed CaS and its coupling via
GÎ±i/o to Ca2+ mobilization and
GÎ±q to PKCÎ± phosphorylation, which could account for
rapid reduction of CaS responses.</p><!><p>DMEM/F-12 (1:1), Hanks Balanced Salt Solution (HBSS), Fura-2/AM,
Pluronic® F-127, penicillin/streptomycin (100X),
heat-inactivated bovine serum, TRIzol reagent, SuperScript™ II RT and
pCR-XL-TOPO vector were from Invitrogen (Carlsbad, CA). 2-Amino-ethyldiphenyl
borate (2-APB), Gö 6976, ionomycin and phorbol-12-myristate-13 acetate
(PMA) were from EMD Biosciences (La Jolla, CA). CaS polyclonal antibody
(PA1-37213), raised against a synthetic peptide corresponding to the N-terminus
of rat CaS and Halt Protease Inhibitor Cocktail were from Pierce Biotechnology
(Rockford, IL). Calindol, rabbit polyclonal PKCÎ± (sc-208) and
p-PKCÎ± (sc-12356-R) antibodies were from Santa Cruz Biotechnology, and
pertussis toxin (PTX) was from Biomol International (Plymouth Meeting, PA). All
other chemicals used were of the purest grade available commercially.</p><!><p>A stable GÎ±q antisense-knockdown (KD) clone was
derived from N18TG2 cells as follows: Cells were transfected (Lipofectamine in
Opti-MEM media) with the full-length 1.7 Kb cDNA coding sequence of
GÎ±q that had been ligated into pcDNA3 (Invitrogen,
Carlsbad, CA) in an antisense orientation (Gardner et al. 2002). Clones were selected by
resistance to G418 sulfate (Mediatech, Herndon, VA) and maintained in media
containing 250 Î¼g/ml G418 sulfate in DMEM/F12 (1:1) medium supplemented
with heat-inactivated bovine serum (10%) and penicillin/streptomycin
(100 U ml-1/100 Î¼g.ml-1). Cells were grown on
glass cover slips for [Ca2+]i
determination.</p><!><p>Reverse transcription-polymerase chain reaction (RT-PCR) was carried out
with total RNA extracted from sub-confluent cells to determine whether N18TG2
cells express mRNA that is homologous with the CaS message expressed in DRG
neurons. The forward primer sequence (5′>GCT
ATA AGC TTC ACT TCT CAG GAC TCG AGG
ACC AGC<3′) is specific for the exon 1 splice variant that is
expressed in DRG but not in the kidney or parathyroid glands, and a reverse
primer sequence (5′>GCT ATG GAT
CCT AAT ACG TTT TCC GTC ACA GAG C<3′) is
based on 3′-UTR sequence that is common in the three tissues.
Hind III and Bam H1 sites (underlined)
were inserted in the forward and reverse primers, respectively, for cloning. The
PCR product was cloned into the pCR-XL-TOPO vector and sequenced with an ABI
Prism 373 Genetic Analyzer (Applied Biosystems, Carlsbad, CA) using M13
forward/reverse primers to establish identity.</p><p>To determine the expression of PKC isoforms, cells were harvested at
90% confluence with Trizol/10% and β-mercaptoethanol,
and lysed using Qiashredder (Qiagen Inc. Valencia, CA). RNA was extracted using
the RNeasy kit (Qiagen Inc. Valencia, CA). RNA concentrations were read on a
Nanodrop 2000 (Thermo Scientific) and cDNA was generated from the RNA having a
260/280 ratio > 1.8 using the First Strand RT2 kit (SA Biosciences
Frederick, MD). PKC isoform expression levels were determined using the mouse
"Human Alzheimer's Disease" RT2
Profiler™ PCR Array Cat # PAMM-057 (Qiagen Inc.
Valencia, CA). ΔCT values were calculated as an average CT from 3 PCR
Array plates minus the mean of the following reference genes, GAPDH,
β-actin and Hsp90abl from the same plates. The ΔΔCT was
determined by comparing each ÎCT to that of N18TG2 WT PKCÎ±, and
the 2-ΔΔCT values were normalized to N18TG2 WT
PKCÎ±, as 100. The data were analyzed by a 2-way ANOVA and the Holm-Sidak
multiple comparisons and Bonferroni tests were used to compare the Nl8TG2 WT
with the GÎ±q KD cells.</p><!><p>Changes in [Ca2+]i in
N18TG2 cells, following stimulation with
[Ca2+]e or calindol, were
determined by microfluorimetric, dual wavelength
[Ca2+]i imaging with Fura-2.
Cells grown on glass cover slips in 35 mm dishes for 48 hr. were loaded with 5
Î¼M Fura-2/AM in HBSS with 0.1% Pluronic for 30 min at 37
°C followed by washing with PSS (mM: NaCl, 150; KC1, 5.4;
MgSO4.7H2O, 1.2; NaH2PO4, 1.2;
NaHCO3, 6.0; CaCl2, 0.25; glucose, 5.5; HEPES, 20; pH
7.4). Cover slips were then mounted in a stainless steel cell chamber
(Attofluor®) in fresh PSS and placed on the stage of an Axiovert 100S
inverted microscope equipped with a Zeiss Fluar 40× oil-immersion
objective. A Dual-wavelength Fluorescence Imaging System (Photon Technology
International, Birmingham, NJ) was used to measure changes in
[Ca2+]i following stimulation
of cells with Ca2+ or calindol, a positive allosteric
modulator of the CaS, in the presence or absence of PTX, 2-APB, nifedipine, PMA
and Gö 6976. Cells loaded with Fura-2 were excited at 340 nm and 380 nm
with a xenon light source (75 Watt Xe Compact Arc Lamp) and emissions at 510 nm
were captured by an IC-300 intensified CCD or CoolSNAP
HQ2 cameras. The images were transmitted to a
computer and processed using the ImageMaster Pro™ or
EasyRatioPro™ Ratio Fluorescence Imaging software, with a macro based on
the concentration equation of Grynkiewicz (Grynkiewicz et al. 1985) The
[Ca2+]i data are reported as
emission ratios (F340/F380). In all experiments, 20-30
cells from a single field were analyzed and the means calculated. Applications
of 10 Î¼M ionomycin followed by 20 mM EGTA were used to determine the
maximum (Rmax) and the minimum
(Rmin) levels of
[Ca2+]i, respectively.</p><!><p>To determine the activation of PKC following stimulation of cells with
increasing concentrations of Ca2+, we analyzed PKCÎ±
and phosphorylated PKCÎ± (p-PKCÎ±) in crude plasma membrane and
supernatant fractions of cell homogenates by Western blotting with PKCÎ±
(sc-208; SCBT) and p-PKCÎ± (sc-12356-R; SCBT) antibodies. Immunoblotting
was carried out on membrane fractions (100K × g pellets) from N18TG2 (WT
and GÎ±q KD) cells. Briefly, cells were grown in 100 mm dishes
for 48 hr. and stimulated with
[Ca2+]e (1 - 5 mM) in the
presence or absence of 1 μM Gö 6976 for 30 min in physiological
salt solution (with 0.25 mM Ca2+). Cells were then scraped
into 12-ml snap-cap tubes and homogenized in Tris buffer (10 mM Tris, pH 7.5;
0.25 M sucrose and 3 mM MgCl2) with freshly-added Halt Protease
Inhibitor Cocktail. The homogenates were passed through a 22-guage needle and
sonicated for 20 sec to complete lysis. Lysates were centrifuged at 800
× g for 10 min at 4 °C and the supernatants centrifuged at
100,000 × g for 1 hr at 4 °C. The supernatants were removed and
pellets suspended in Spiegel's buffer (20 mM Tris, pH 6.8; 150 mM NaCl;
and 10 mM EDTA; 1% Triton X-100; 1 mM EGTA) with freshly-added Halt
Protease Inhibitor Cocktail. Protein concentrations of the supernatants and
pellets were determined by the BCA method and 100 Î¼g samples were
separated by electrophoresis on 8% sodium dodecyl sulfate polyacrylamide
gel and transferred onto PVDF membranes. The membranes were then blotted with
PKCÎ± or p-PKCÎ± antibodies, incubated with horseradish
peroxidase-conjugated secondary IgG, and visualized using Enhanced
Chemiluminescence (ECL™).</p><!><p>Data were analyzed with SigmaPlot 11.0 statistics programs from Systat
Software, Inc. (Point Richmond, CA). Comparisons between groups and within
groups were done by One Way Analysis of Variance (ANOVA);
differences with p < 0.05 were considered
significant.</p><!><p>The RT-PCR product (Figure 1A),
cloned into the pCR-XL-TOPO vector, was sequenced and the BLAST data obtained
from sequences in GeneBank database, indicate 99.7% sequence identity
(578 of 580 bases; 2 mismatches) between the N18TG2 CaS cDNA sequence and mouse
(C57BL/6J strain) genomic CaS sequence (GI: 4731165) in chromosome 1,
97.9% identity (550 of 562 bases; 8 mismatches) with the Norway rat
genomic CaS sequence (GI: 8393053) in chromosome 11, and 88.5% identity
(323 of 365 bases; 1 mismatch) with the human genomic CaS sequence (GI: 904210)
in chromosome 3. Western blot analysis of proteins extracted from N18TG2 cells
showed levels of expression of a protein of size (≈ 150 kDa), comparable
to the DRG and parathyroid CaS (Fig. 1B).
In order to investigate signal transduction that occurs via Gq, we developed a
stable anti-sense GÎ±q KD clone which expresses about
33% (calculated from the Western blot densitometry data) less
GÎ±q protein subunit (Fig
1C). Of note, WT and GÎ±q KD cells express similar
amounts of CaS.</p><!><p>To characterize CaS-mediated changes in
[Ca2+]i in N18TG2 cells, 1 mM
Ca2+e or 5 Î¼M calindol was applied to
Fura-2-loaded cells on glass cover slips (Fig.
2). The change in
[Ca2+]i consisted of an
initial rise followed by a gradual decline to plateau above basal. The rate of
increase to peak [Ca2+]i with 1 mM
Ca2+ was higher in GÎ±q KD compared to
WT cells, but the differences were not statistically significant. The CaS in
N18TG2 cells responded to either 5 Î¼M or 10 Î¼M calindol in the
presence of 0.25 mM Ca2+, a concentration that serves as the
background level in the present studies (Fig.
2C-E). Both WT and GÎ±q KD cells responded with
robust, concentration-dependent increases in Ca2+
mobilization, which rose at a higher rate than those observed after stimulation
of CaS by 1 mM Ca2+. The changes in
F340/F380 ratios for 10 Î¼M calindol were: WT
cells, 1.32 ± 0.14 vs. Gαq KD cells, 1.57 ±
0.18. Compared with the response to Ca2+, the response to
calindol continued for a longer period and declined to a plateau that was
greater than the background level for both WT and GÎ±q KD
cells.</p><!><p>In order to assess contributions of GÎ±q versus
GÎ±i/o in Ca2+ mobilization in N18TG2
cells, we examined the effects of inactivating GÎ±i/o proteins
with PTX on Ca2+ and calindol responses. If
GÎ±q were contributing to the Ca2+
mobilization, one would predict a lower peak value in the GÎ±q
KD cells. Rather, Fig. 3 shows that
pre-treatment of both N18TG2 WT and GÎ±q KD cells with 50
ng/ml PTX overnight reduced responses to 1 mM Ca2+ by 68
± 2 % in WT cells (n = 4, p <
0.05 vs. controls) and by 82 ± 2% (n = 4;
p < 0.01) in GÎ±q KD cells (Fig. 3A-C). Calindol-induced peak responses
in WT cells were reduced by 35 ± 2 % with no effect on the
plateau. In GÎ±q KD cells, peak Ca2+ was
reduced by 60 ± 3% and the plateau by 35 ± 3% (n
= 4-6) (Fig. 3D,E). Notably, the
diminished levels of GÎ±q in the GÎ±q KD
cells did not further reduce the rate of rise to peak compared with WT in the
GÎ±i/o-inactivated cells. These data demonstrate that
Ca2+ mobilization is under the dominant regulation of
GÎ±i/o proteins.</p><!><p>To examine the role of the IP3-responsive channel in
Ca2+ signaling by the CaS in N18TG2 cells, we treated the
cells with an IP3 and SOCE channel inhibitor, 2-APB (30 Î¼M)
for 20 min and measured the level of
[Ca2+]i. Fig. 4A.-C. shows that 2-APB eliminated the
Ca2+-stimulated rise to a peak
[Ca2+]i in WT, and
significantly reduced the peak by 78 ± 2% (n = 4;
p < 0.001 vs. control) in GÎ±q KD
cells. To determine if L-type dihydopyridine-sensitive Ca2+
channels play a role in the neuronal CaS-mediated Ca2+
influx, we examined the effect of nifedipine on Ca2+
mobilization in WT and GÎ±q KD cells. Cells were treated with
50 Î¼M nifedipine or vehicle for 20 min and
[Ca2+]i measured in response
to a 1 mM Ca2+ stimulus. Fig. 4
D.-F. shows that the rate of rise to peak
[Ca2+]i was attenuated
following pre-treatment with nifedipine for both WT and GÎ±q
KD cells. The rate of rise, determined as the change in initial slope of
tracings over time, was more gradual at 4.7 × 10-3 units/sec
for nifedipine-treated cells vs.15.1 × 10-3 units/sec for
control, a 3-fold reduction. The peak
[Ca2+]i heights were reduced
by about 50% in both cell types. Plateau levels of
[Ca2+]i were the same in the
presence or absence of nifedipine in both WT and GÎ±q KD
cells.</p><!><p>Ca2+i mobilization is likely to activate
PKC as a primary mechanism of signaling. As shown in Fig. 5, N18TG2 cells express abundant mRNA for
PKCÎ±, PKCÎ´, PKCÎµ, PKCÏ, and PKCÎ¶
isoforms (for review of PKC sub-families and their activation, see (Olive & Newton 2010, Wu-Zhang & Newton 2013). Of the
conventional PKC isoforms that translocate to the plasma membrane in response to
[Ca2+]i and diacylglycerol,
PKCÎ± was expressed in 100-fold greater abundance than PKCÎ² or
Î³. The PKCÎ± mRNA levels were not changed by knock-down of
GÎ±q (Fig. 5) or by
the over-night treatment with PTX to block Gi/o stimulation (data not
shown). The 2-way ANOVA indicated significant differences between the PKC
subtypes as well as between the cell lines (p <
0.0001), with a significant interaction between these factors
(p < 0.002). Significant differences (p
< 0.05) were observed between cell lines for Î´,
τ and ζ, PKC subtypes.</p><p>To determine the role played by conventional PKC in CaS-mediated
Ca2+i mobilization in N1 8TG2 cells, we
incubated the cells with PMA, an activator of PKC that mimics the diacylglycerol
required for membrane association (Fig. 6).
Pre-treatment of cells with 100 nM PMA for 20 min reduced CaS-mediated
Ca2+i mobilization by 73 ± 2%
(n = 4, p < 0.01 vs. control) in N18TG2 cells
(Fig. 6A,C) and 79 ± 4%
(n = 4; p < 0.01) in GÎ±q KD
cells (Fig. 6B,C). The plateau
[Ca2+]i was not affected. We
also determined the effect of the Ca2+i -specific
PKC inhibitor, Gö 6976 (a cell-permeable, reversible and
ATÎ²-competitive inhibitor of PKCÎ± and PKCÎ²) on
Ca2+ mobilization. In both cell types, 100 nM Gö
6976 had no effect on peak Ca2+ but reduced the amplitudes
(Fig. 7A, B). This concentration also
reduced the plateau [Ca2+]i in
GÎ±q KD cells (Fig.
7B). However, 1 μM Gö 6976 reduced the peak and
plateau [Ca2+]i in both cell
types. The plateau was completely eliminated in GÎ±q KD cells.
In the current study, we focused on PKCÎ± to investigate activation and
translocation in response to CaS signaling.</p><p>To further investigate the mechanism by which PKC regulates the neuronal
CaS response, we determined the effects of increasing
[Ca2+]e on PKC phosphorylation
and translocation. Western blot analysis indicated that membrane-associated
PKCÎ± (Figs. 9A,B) and p-PKCÎ±
(Figs. 9C,D) levels increased when
[Ca2+]e was raised from the
basal level of 0.25 mM to 5 mM in WT cells. In contrast, GÎ±q
knockdown increased PKCÎ± (Fig. 8B)
and p-PKCÎ± (Fig. 8D) levels in
basal conditions (0.25 mM Ca2+) and reversed the PKCÎ±
phosphorylation and translocation patterns in response to increased
[Ca2+]e. Upon pretreatment
with Gö6976, Ca2+e failed to increase
membrane-associated PKCÎ± and p-PKCÎ± levels. These findings
indicate that GÎ±q signaling directs the PKCÎ± cellular
response. Inhibition of PKC by Gö 6976 reduced the
[Ca2+]e-dependent increase and
decrease in membrane-associated PKCÎ± and p-PKCÎ± levels.</p><!><p>CaS activation leads to signal transduction through cyclic AMP and
phospholipases depending on the cell type (Awumey et
al. 2007, Brown & MacLeod
2001, Wang et al. 2003a, Wang et al. 2003b). Activation of PLC leads to production of
IP3R-mediated release of stored Ca2+ from the ER
causing a transient rise in [Ca2+]i.
ER Ca2+ store emptying also opens plasma membrane SOCE channels
that allow influx of Ca2+ resulting in a sustained plateau as
shown in the present study.</p><p>In the present study we investigated the effects of
[Ca2+]e on
[Ca2+]i signal transduction via
GÎ±i/o and GÎ±q in mouse N18TG2, a neuronal
cell model that endogenously express a CaS having > 90% homology
with the rat and human neuronal CaS receptors. In other tissues, the CaS exerts its
effects mainly by interacting with GÎ±q, GÎ±i/o
and GÎ±12/13 (Brown &
MacLeod 2001, Ward 2004). Our
studies indicate that the neuronal CaS requires a signaling pathway mediated by
PTX-sensitive Gi/o proteins for Ca2+ mobilization. In
parathyroid tissue, CaS was reported to transduce the mobilization of
Ca2+i through GÎ±i/o proteins,
with PI-PLC as the major downstream effector (Fitzpatrick et al. 1986, Brown et al. 1993). Stimulation of PI-PLC in AtT-20
pituitary cells (Emanuel et al.
1996), and Xenopus oocytes (Brown et al. 1993) expressing exogenous CaS also occurred
via PTX-sensitive proteins. In contrast, bovine parathyroid cell CaS couples to
PI-PLC through Gq/11 (Brown 1999).
The CaS exogenously expressed in HEK293 cells is also coupled to
GÎ±q/11 (Kifor et
al. 1997).</p><p>Our data also indicate that the CaS-mediated Ca2+
mobilization is sensitive to nifedipine suggesting involvement of
dihydropyridine-sensitive Ca2+ channels. This is consistent with
a report indicating the presence of nifedipine-sensitive Ca2+
channels and N-type Ca2+ channels in a similar neuronal cell line
(Schneider et al. 1995).
However, the plateau was not affected, indicating that nifedipine may affect the
kinetics of release of Ca2+ from ER and influx. In neutrophils,
nifedipine inhibits Ca2+ release from intracellular stores (Rosales & Brown 1992). Furthermore,
dihydropyridine-sensitive L-type Ca2+ channels influenced
IP3-induced Ca2+ release in angiotensin
II-stimulated glomerulosa cells (Spat et
al. 1996), and nifedipine completely blocked slow
Ca2+ transients in primary cultures of rat myotubes (Araya et al. 2003). ER
Ca2+ stores play an important role in increasing
[Ca2+]i in neuronal cells (Berridge 2004, Putney 2005), although voltage-dependent Ca2+
channels, plasma membrane Ca2+-ATPase, SOCE and the
Na+/Ca2+ exchanger also play critical
roles in the influx and recovery of resting
[Ca2+]i in rat sensory neurons
Thus, the effect of nifedipine may be much more complex that is apparent in the
present study.</p><p>The present data demonstrate that in N18TG2 cells, Ca2+
mobilization is up-regulated in GÎ±q KD cells suggesting that
GÎ±q is not intrinsic to the Ca2+
mobilization mechanism, but is important to the modulation of the response. The CaS
has numerous phosphorylation sites in the extracellular domain thus, our findings
that PMA reduced the CaS-mediated responses, and that the reduced response is
accompanied by PKC phosphorylation and mobilization, as well as the reduction of
Ca2+ mobilization response and membrane translocation by
Gö 6976, provide evidence for an alternative mechanism to regulate the
sensitivity of the neural CaS. The reduction in Ca2+ mobilization
(Ca2+ peak, amplitude and plateau) by the higher
concentration of Gö 6976 in both cell types suggests the involvement of
other Ca2+ mobilization pathways, such as the ER
Ca2+-ATPase and store-operated Ca2+ entry
channels. The effect of PKC inhibition on Ca2+ mobilization in
the present study is similar to that reported for other PKC inhibitors (Bis-I, Ro
31-8220, Ro 32-0432) and GÓ§ 6976 in HEK293 cells expressing the DRG CaS-EGFP
fusion protein (Awumey et al.
2007). The Ca2+ influx pathway (the plateau) was
blocked in GÎ±q KD cells, thus, PKC inhibition shortened the
duration of Ca2+ mobilization in these cells suggesting that the
inhibitor effect is on both the rate of Ca2+ release from ER and
Ca2+ influx that appears to be much more complex and requires
further investigation.</p><p>These studies suggest that activated PKC can suppress the
Ca2+i mobilization response to CaS stimulation in
both WT and GÎ±q KD cells. Presumably, PMA treatment of cells
resulted in the activation of PKC and subsequent phosphorylation of the CaS leading
to reduced Ca2+ mobilization. PKC is known to phosphorylate a
critical threonine in the human parathyroid CaS, and this phosphorylation modulates
the functional interaction with G proteins (Bai
et al. 1998) critical to the regulation of
Ca2+ release from internal stores (Awumey et al. 2007, Jiang
et al. 2002). The present findings demonstrate a
role for [Ca2+]e-induced translocation
and phosphorylation of PKC in modulation of the neural CaS response, which was
protected by diminished GÎ±q levels in the cell, as was the
phosphorylation and mobilization of PKC to the membrane, indicating that
GÎ±q is intrinsic to the modulation of CaS signaling, rather
than the Ca2+ mobilization mechanism.</p><p>We conclude that in N18TG2 neuroblastoma cells, which endogenously express
the CaS with native GÎ±i/o and GÎ±q, stimulation
with extracellular Ca2+ and the allosteric modulator, calindol
promotes rapid increases in [Ca2+]i,
which is mediated by GÎ±i/o. Furthermore, Ca2+
and diacylglycerol-activated PKCÎ± pathway modulates the CaS as a result of
PKC phosphorylation and translocation to the membranes following activation by
agonist. Our finding that this process is down-regulated in the
GÎ±q KD cells indicates a functional requirement for
GÎ±q in mediating the PKC influence on CaS responses. The
current study provide the impetus to seek novel agonists or positive allosteric
regulators biased toward coupling the CaS to either GÎ±i/o to
sustain an activated state, or GÎ±q to promote reduced responses.
That an established neural-derived cell line expresses a CaS, provides a potentially
useful model system to screen for compounds that are selective in cellular
regulation of CaS-mediated responses.</p>