Role of Ca2+ Feedback on Single Cell Inositol 1,4,5-Trisphosphate Oscillations Mediated by G-protein-coupled Receptors*
Kenneth W. Young
,
Mark S. Nash,
R. A. John Challiss and
Stefan R. Nahorski
From the
Department of Cell Physiology and Pharmacology, Medical Sciences
Building, University of Leicester, University Road, Leicester LE1 9HN, United
Kingdom
Received for publication, November 13, 2002
, and in revised form, March 10, 2003.
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ABSTRACT
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The dynamics of inositol 1,4,5-trisphosphate (Ins (1,4,5)P3)
production during periods of G-protein-coupled receptor-mediated
Ca2+ oscillations have been investigated using the
pleckstrin homology (PH) domain of phospholipase C (PLC)
1
tagged with enhanced green fluorescent protein
(eGFP-PHPLC
1). Activation of
noradrenergic
1B and muscarinic M3 receptors
recombinantly expressed in the same Chinese hamster ovary cell indicates that
Ca2+ responses to these G-protein-coupled receptors are
stimulus strength-dependent. Thus, activation of
1B
receptors produced transient base-line Ca2+
oscillations, sinusoidal Ca2+ oscillations, and then a
steady-state plateau level of Ca2+ as the level of
agonist stimulation increased. Activation of M3 receptors, which
have a higher coupling efficiency than
1B receptors,
produced a sustained increase in intracellular Ca2+ even
at low levels of agonist stimulation. Confocal imaging of
eGFP-PHPLC
1 visualized periodic
increases in Ins(1,4,5)P3 production underlying the base-line
Ca2+ oscillations. Ins(1,4,5)P3 oscillations
were blocked by thapsigargin but not by protein kinase C down-regulation. The
net effect of increasing intracellular Ca2+ was
stimulatory to Ins(1,4,5)P3 production, and dual imaging
experiments indicated that receptor-mediated Ins(1,4,5)P3
production was sensitive to changes in intracellular
Ca2+ between basal and
200 nM. Together,
these data suggest that
1B receptor-mediated
Ins(1,4,5)P3 oscillations result from a positive feedback effect of
Ca2+ onto phospholipase C. The mechanisms underlying
1B receptor-mediated Ca2+ responses
are therefore different from those for the metabotropic glutamate receptor 5a,
where Ins(1,4,5)P3 oscillations are the primary driving force for
oscillatory Ca2+ responses (Nash, M. S., Young, K. W.,
Challiss, R. A. J., and Nahorski, S. R. (2001) Nature 413,
381382). For
1B receptors the
Ca2+-dependent Ins(1,4,5)P3 production may
serve to augment the existing regenerative Ca2+-induced
Ca2+-release process; however, the sensitivity to
Ca2+ feedback is such that only transient base-line
Ca2+ spikes may be capable of causing
Ins(1,4,5)P3 oscillations.
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INTRODUCTION
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Frequency encoding of Ca2+ signals in the form of
oscillations provides a versatile mechanism by which a number of intracellular
processes are controlled (1,
2,
3). Intracellular
Ca2+ oscillations, which arise from periodic release
from intracellular stores, can be manifest as either transient oscillations
from a base-line Ca2+ level or sinusoidal oscillations
upon a raised plateau level of Ca2+ (see Refs.
4 and
5). Putative mechanisms by
which cell surface G-protein-coupled receptors
(GPCRs)1 stimulate
Ca2+ oscillations via the intracellular messenger
molecule Ins(1,4,5)P3 can be broadly split into two categories,
dependent on whether levels of Ins(1,4,5)P3 are required to
oscillate (6,
7). Thus,
Ca2+ oscillations driven by a steady state raised level
of Ins(1,4,5)P3 may occur via a regenerative
Ca2+-induced Ca2+-release process
(CICR), which is an integral property of the Ins(1,4,5)P3 receptor
(InsP3R) (see Ref.
5). In contrast, the
sensitivity of certain oscillatory Ca2+ responses to
feedback inhibition by protein kinase C (PKC), or regulators of G-protein
signaling (RGS) proteins, suggests that Ins(1,4,5)P3 levels may
oscillate and hence drive Ca2+ oscillations
(8,
9,
10,
11). These two categories need
not be mutually exclusive as, for example, Ca2+ may be
required to recruit PKC to the plasma membrane before feedback inhibition can
occur (12).
The recent introduction of the pleckstrin homology (PH) domain of
phospholipase C-
1 tagged with enhanced green fluorescent
protein (eGFP-PHPLC
1) to detect
Ins(1,4,5)P3 in single cells
(13,
14,
15) has enabled the profile of
agonist-stimulated Ins(1,4,5)P3 production to be investigated
directly. This method has indeed detected Ins(1,4,5)P3 oscillations
in ATP-stimulated Madin-Darby canine kidney cells
(14) and metabotropic
glutamate receptor 5a (mGluR5a)stimulated CHO cells
(10,
12). Ins(1,4,5)P3
oscillations mediated by mGluR5a activation arise from a PKC-dependent dynamic
uncoupling of the receptor from its signaling pathway
(9,
10,
12) giving rise to synchronous
Ins(1,4,5)P3 and Ca2+ oscillations. In
contrast, however, muscarinic M3 receptor-driven sinusoidal
Ca2+ oscillations in CHO cells were associated with a
modest steady-state increase in Ins(1,4,5)P3
(10).
This current study has investigated the cellular dynamics of
Ins(1,4,5)P3 production and Ca2+ signaling in
individual CHO cells in response to noradrenergic
1B and
muscarinic M3 receptor stimulation, using the mGluR5a model for
PKC-dependent dynamic receptor uncoupling as a comparison. Our results provide
the first direct evidence that Ca2+ oscillations may
stimulate transient increases in Ins(1,4,5)P3 via a positive
feedback effect on phospholipase C (PLC). Moreover, although PKC can regulate
receptor activity, it appears not to be directly involved in the
Ins(1,4,5)P3 oscillations initiated by
1B
receptors. Furthermore, the Ca2+ sensitivity of PLC
isoforms in CHO cells is such that only Ca2+
oscillations of the transient base-line spiking nature would be capable of
stimulating this positive feedback. In comparison, in the same cells,
mGluR5a-mediated Ins(1,4,5)P3 oscillations appear to contain
elements of both positive Ca2+ feedback and dynamic
PKC-mediated inhibition, dependent on the agonist concentration. The role of
this Ca2+ feedback-induced Ins(1,4,5)P3
production may be to enhance the regenerative CICR process by increasing the
number of Ins(1,4,5)P3-bound InsP3Rs available for
activation.
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MATERIALS AND METHODS
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Cell Culture and Plasmid TransfectionCHO cells stably
expressing noradrenergic
1B (2.3 pmol/mg protein) and
muscarinic M3 receptors (3.4 pmol/mg protein)
(CHO
1B/M3) were maintained in minimum Eagle's
medium-
(Invitrogen) supplemented with 10% fetal calf serum, 2 mM
L-glutamine, and 2.5 µg/ml fungizone, 400 µg/ml G418, and 300
units/ml hygromycin. Metabotropic glutamate receptor 5a expressing CHO cells
(CHO-lac-mGlu5a) were created using the LacSwitch IIinducible expression
system (Stratagene) as described previously
(12). Maximal levels of
mGluR5a expression were achieved by preincubation with 100 µM
isopropyl-1-thio-
-D-galactopyranoside
(12). Cells were plated to
near confluence on coverslips before being transfected with 1 µg of plasmid
DNA plus 3 µl of FuGENE 6 (Roche Applied Science) per coverslip (according
to manufacturer's instructions). Cells were used 48 h after transfection. For
experiments examining mGluR5a transiently expressed in
CHO
1B/M3 cells, 0.5 µg of mGluR5a in pcDNA3
(Invitrogen) was co-transfected with 0.5 µg of
eGFP-PHPLC
1 with 3 µl of FuGENE 6
per coverslip. The medium was changed after 24 h to prevent accumulation of
extracellular glutamate, and cells were used after a further 24 h. The
eGFP-PHPLC
1 construct was a kind gift
from Prof. Tobias Meyer.
Single Cell Imaging of Ins(1,4,5)P3 and
Ca2+Coverslips were
mounted on the stage of an Olympus IX70 inverted epifluorescence microscope
and perfused at 37 °C with Krebs-Henseleit buffer (in mM: NaCl
118, KCl 4.7, MgSO4 1.2, CaCl2 1.3,
KH2PO4 1.2, NaHCO3 4.2, HEPES 10, glucose
11.7, pH 7.4). Images of cells after excitation at 488 nm were collected using
an Olympus FV500 laser scanning confocal microscope at a scan rate of
1.52.5 Hz. Increases in cellular Ins(1,4,5)P3 were detected
by measuring the translocation of
eGFP-PHPLC
1 from the plasma membrane
to the cytosol. This was done by creating a cytosolic region of interest and
plotting the average pixel intensity in that region versus time. Data
are expressed in relative fluorescent units (RFU) by subtraction of background
fluorescence followed by dividing the fluorescent intensity at a given time by
the initial fluorescence within each region of interest
(F/F0). Experiments involving co-detection of
Ins(1,4,5)P3 and Ca2+ were made by
transfecting CHO
1B/M3 cells with
eGFP-PHPLC
1 as above and then loading
the cells with fura-2 (2 µM fura-2AM, 1 h). The cells were then
excited at 340 and 380 nm (for fura-2) and 488 nm (for eGFP) using a
Spectramaster II monochromator (PerkinElmer Life Sciences). Images were
collected via a cooled CCD camera using the Merlin2000 data acquisition system
(PerkinElmer Life Sciences) at a sample rate of 0.7 Hz. Changes in cytosolic
eGFP-PHPLC
1 were measured as described
above. Simultaneously, changes in cytosolic Ca2+
([Ca2+]i) were measured by
converting the 340/380 ratio of fluorescence (after background subtraction) to
approximate [Ca2+]i using the method
of Grynkiewicz et al.
(16). The minimal and maximal
fluorescence ratios (Rmin and Rmax)
were obtained from a sample set of CHO cells using 5 µM
ionomycin and 6 mM EGTA (for Rmin) followed by
10 mM CaCl2 (for Rmax).
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RESULTS
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Receptor-dependent Ca2+
Oscillations in CHO CellsThe temporal patterns of
intracellular Ca2+ signals in CHO cells co-expressing
noradrenergic
1B and muscarinic M3 receptors were
compared with those elicited by the metabotropic glutamate receptor mGluR5a.
The
1B receptor was capable of stimulating
Ca2+ oscillations over a range of agonist
concentrations. Thus, increasing the concentration of noradrenaline (NA) from
10-8 to 3 x 10-7
M produced base-line Ca2+ oscillations, the
frequency of which increased with the stimulus strength
(Fig. 1A). At NA
concentrations above 10-6 M a steady-state
plateau level of Ca2+ was observed
(Fig. 1A). In
contrast, although oscillatory Ca2+ responses were
occasionally observed at low agonist doses (data not shown)
(10), in most cells examined,
concentrations of methacholine (MCH) of 10-8
M and above produced a peak and plateau Ca2+
response (Fig. 1B).
Oscillatory Ca2+ signals in CHO-lac-mGlu5a cells,
occurring via cyclical changes in Ins(1,4,5)P3 production
(12), differed from those
observed with either NA or MCH in CHO
1B/M3 cells,
in that they were largely of constant frequency and did not saturate with
increasing agonist concentration. Thus at the lowest concentration of
glutamate investigated (L-Glu, 10-6
M), base-line Ca2+ transients could be
observed (Fig. 1C).
Increasing the concentration of L-Glu to 3 x
10-6 M increased the frequency of these
base-line oscillations, but further increasing the concentration of
L-Glu even up to 3 x 10-4 M
had no additional effects on the Ca2+ signal
(Fig. 1C).

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FIG. 1. Receptor-dependent Ca2+ oscillations in CHO
cells. CHO 1B/M3 (A and B)
and CHO-lac-mGlu5a (C) cells were loaded with fura-2AM, and single
cell images of changes in intracellular free Ca2+
concentration ([Ca2+]i) were
measured on an inverted epifluorescence microscope. Each concentration of
agonist, NA (A), MCH (B), and L-Glu (C),
was applied for 200 s. Graphs are sample traces from more than 45
cells. Ratios of fluorescence
F340/F380 were converted to
[Ca2+]i as described under
"Materials and Methods."
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Receptor-dependent Ins(1,4,5)P3
Oscillations in CHO Cells Simultaneous single cell
measurements of Ins(1,4,5)P3 production (using
eGFP-PHPLC
1) and
Ca2+ mobilization (using fura-2) demonstrated that
although the
1B and M3 receptors are co-expressed
at similar levels in the CHO
1B/M3 cells, the
muscarinic M3 receptor stimulates
10-fold more
Ins(1,4,5)P3 production at maximal agonist concentrations
(Fig. 2A). In
contrast, due to the large degree of amplification between
Ins(1,4,5)P3 production and subsequent Ca2+
mobilization (17), the peak
Ca2+ responses produced by NA and MCH were not
significantly different (Fig.
2A, inset, measured simultaneously from the same
cells as Fig. 2A,
main panel). When temporal changes in Ca2+ and
Ins(1,4,5)P3 were co-imaged, small oscillatory changes in
Ins(1,4,5)P3 were observed during periods of NA-induced base-line
Ca2+ spiking (Fig.
2B). However, due to the limited sensitivity of this
method for detecting cytoplasmic changes in
eGFP-PHPLC
1, further experiments were
conducted using confocal microscopy.

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FIG. 2. Co-imaging of Ins(1,4,5)P3 and
[Ca2+]i in
CHO 1B/M3 cells. Cells were transfected with
eGFP-PHPLC 1 for 48 h before being
loaded with fura-2AM. A, concentration-response data for increases in
Ins(1,4,5)P3 (RFU) and
[Ca2+]i
(F340/F380) were measured simultaneously from the same
cells with increasing concentrations of agonist (NA, filled circles)
or MCH (open circles) separated by a 2-min wash period. Data are the
mean ± S.E. from 5 to 7 cells. A, inset, mean peak
Ca2+ increase in response to 10-4
M MCH (light column) and 10-4
M NA (dark column) as measured from the same cells in main
panel of A. B, cells were co-imaged as above during a prolonged
application of 10-7 M NA. Initial stimulation
with 10-7 M NA produced a small raised
plateau level of Ins(1,4,5)P3 and a Ca2+
response that approximated to sinusoidal oscillations. At later time points
base-line Ca2+ spiking was observed, and simultaneous
with these were small transient increases in intracellular
Ins(1,4,5)P3. Trace is representative of three cells in which
base-line Ca2+ spiking and simultaneous
Ins(1,4,5)P3 oscillations were observed.
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Confocal imaging more clearly revealed the presence of an oscillatory
Ins(1,4,5)P3 response to low concentrations of NA in
CHO
1B/M3 cells. This was most prevalent with
10-7 M NA, which was around the threshold
level for Ins(1,4,5)P3 detection
(Fig. 3B). Oscillatory
Ins(1,4,5)P3 signals in response to 10-7
M NA were characterized by a larger initial peak of
Ins(1,4,5)P3 production (increase in
F/F0 value of 0.40 ± 0.05 RFU, mean of 47
cells), followed by smaller transient increases of relatively consistent
height (0.15 ± 0.01 RFU increase, 141 oscillations in 47 cells,
measured during periods of base-line Ins(1,4,5)P3 oscillations).
The average frequency of the Ins(1,4,5)P3 oscillations in response
to 10-7 M NA was 0.042 ± 0.003 Hz, or
1 peak every 23.6 s (47 cells). Increasing the concentration of NA altered the
temporal dynamics of response, such that at 10-4
M NA a peak and plateau increase in Ins(1,4,5)P3 was
observed (0.64 ± 0.09 RFU initial peak increase, and 0.35 ± 0.04
RFU increase after 120 s, 8086 cells)
(Fig. 3A). As already
described above, the M3 receptor is more efficiently coupled to
Ins(1,4,5)P3 production. In confocal measurements, the most common
temporal profile of Ins(1,4,5)P3 production in response to MCH was
a robust peak and plateau response (Fig.
3C). At 10-4 M MCH this
corresponded to a 6.17 ± 0.28 RFU initial increase in
F/F0, dropping to 4.93 ± 0.24 RFU increase
after 120 s (39 cells). It should be noted that the greater level of
stimulation observed, compared with the co-imaging data, is due to the lower
initial cytosolic fluorescence in the confocal section. On rare occasions,
small oscillatory responses were also observed with MCH stimulation
(Fig. 3D). In three
cells in which M3 receptor-mediated Ins(1,4,5)P3
oscillations were observed, the initial peak increase (0.16 ± 0.01 RFU)
was smaller than that observed with 10-7 M
NA, as were the subsequent peaks (0.10 ± 0.01 RFU increase, 9
oscillations in three cells). The average frequency of these oscillations was
0.032 ± 0.001 Hz, or 1 every 31.2 s.

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FIG. 3. Temporal profile of Ins(1,4,5)P3 responses in
CHO 1B/M3 cells.
CHO 1B/M3 cells were transfected with
eGFP-PHPLC 1, and changes in
Ins(1,4,5)P3 were measured via confocal microscopy. A,
10-4 M NA produced an initial peak of
Ins(1,4,5)P3 production followed by a raised plateau level of
Ins(1,4,5)P3 that was maintained until agonist washout. B,
oscillatory Ins(1,4,5)P3 response after stimulation with
10-7 M NA (representative of 47 cells).
C, a robust peak and plateau Ins(1,4,5)P3 response was
observed with 10-4 M MCH. D,
oscillations in Ins(1,4,5)P3 levels were observed in three cells in
response to stimulation with 10-8 M MCH. Note
the scale bar for C, indicating the greater magnitude of MCH
response.
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Role of Ca2+ and PKC in Single
Cell Ins(1,4,5)P3
OscillationsNA-induced Ins(1,4,5)P3 and
Ca2+ oscillations appeared dependent on both
mobilization of Ca2+ from intracellular stores (as they
were inhibited by addition of the smooth endo-plasmic reticulum
Ca2+ ATPase (SERCA) pump inhibitor, thapsigargin (5
x 10-6 M),
Fig. 4B), and protein
kinase activity (as they were inhibited by staurosporine
(10-6 M),
Fig. 4C). In both
cases, these inhibitors produced raised steady-state levels of
Ins(1,4,5)P3 and [Ca2+]i
in CHO
1B/M3 cells stimulated with
10-7 M NA
(Fig. 4).

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FIG. 4. Role of Ca2+ and PKC on single cell
Ins(1,4,5)P3 oscillations. Left panels,
CHO 1B/M3 cells were transfected with
eGFP-PHPLC 1 and imaged on the confocal
microscope after 48 h. Right panels, in a separate set of
experiments, CHO 1B/M3 cells were loaded with
fura-2AM and changes in [Ca2+]i
imaged on an inverted epifluorescence microscope. A,
10-7 M NA was bath applied to the cells for
400 s, as indicated by the dotted line. Drug was removed from the
coverslip chamber via the perfusion system. B,
Ins(1,4,5)P3 oscillations were initiated by bath application of
10-7 M NA (dotted line).
Thapsigargin (thaps, 5 x 10-6
M final concentration) was added to the coverslip chamber at 300 s,
in the continued presence of 10-7 M NA, as
indicated by the arrow (representative of 10 cells for
Ins(1,4,5)P3 and 17 cells for Ca2+).
C as B, except 10-6 M
staurosporine (stauro) was added at 300 (representative of 99 cells
for Ins(1,4,5)P3 and 114 cells for Ca2+). The
drop in Ins(1,4,5)P3 and
[Ca2+]i toward the end of the
experiment in C is due to the removal of NA and staurosporine via the
perfusion system.
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To investigate further the role of
[Ca2+]i in NA-mediated
IP3 production, CHO
1B/M3 cells were
pretreated with thapsigargin (2 x 10-6
M) in the absence of extracellular Ca2+
(nominally Ca2+ free) for 5 min. Cells were then
stimulated with NA (10-4 M for 200 s) and
then washed in normal KHB (containing 1.3 mM CaCl2) for
10 min, before being stimulated with NA (10-4
M for 200 s) again. In the absence of extracellular
Ca2+, NA produced an initial peak 0.59 ± 0.07 RFU
increase in Ins(1,4,5)P3 levels. This response declined to a near
steady-state plateau phase of Ins(1,4,5)P3 production, consisting
of a 0.20 ± 0.03 RFU increase by 200 s (28 cells)
(Fig. 5, A and
B, plateau phase). After the 10-min wash period
in 1.3 mM Ca2+, NA produced an initial peak
Ins(1,4,5)P3 response that was only marginally greater than that
observed in the absence of extracellular Ca2+ (0.68
± 0.09 RFU increase, 28 cells). However, the plateau phase of
Ins(1,4,5)P3 production, measured as a 0.40 ± 0.07 RFU
increase at 200 s, was double that observed in nominally
Ca2+-free conditions (p = 0.02, paired
t test). Hence it would appear that the net effect of increases in
[Ca2+]i are stimulatory to
Ins(1,4,5)P3 production. It was of interest to note the same
experiment, when conducted on CHO-lac-mGlu5a cells, produced the opposite
result. Thus, mGluR5a stimulation after thapsigargin pretreatment was enhanced
upon Ca2+ removal and reduced by
Ca2+ readdition
(12).

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FIG. 5. Effect of Ca2+ removal on
1B receptor-mediated Ins(1,4,5)P3 production.
A, eGFP-PHPLC 1-transfected
cells were pretreated with thapsigargin (2 x 10-6
M, 5 min) in nominally Ca2+-free KHB before
being stimulated with NA (10-4 M, 200 s) in
nominally Ca2+ free KHB (dotted line). Cells
were then perfused with KHB containing 1.3 mM CaCl2 for
10 min before being stimulated with NA (10-4
M, 200 s) in the presence of 1.3 mM CaCl2
(solid line). B, mean data from experiments described in
A. Removal of extracellular Ca2+ significantly
reduced the plateau phase of NA-mediated Ins(1,4,5)P3 production as
measured at 200 s (p = 0.02, paired t test, n =
28).
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The effect of thapsigargin suggested that a positive feedback effect of
Ca2+ on PLC activity could be the mechanism behind the
observed NA-mediated Ins(1,4,5)P3 oscillations. To determine the
dynamic range of this Ca2+-sensitive
Ins(1,4,5)P3 production, Ca2+ and
Ins(1,4,5)P3 levels in response to 10-4
M NA were measured simultaneously, while extracellular
Ca2+ was stepped from 1.3 to 1.0, 0.3, nominally
Ca2+-free (0 Ca2+), and then back
to 1.3 mM (Fig. 6).
In this set of experiments, 10-4 M NA in the
presence of 1.3 mM extracellular Ca2+
produced peak and plateau Ins(1,4,5)P3 and
Ca2+ responses. Although changing extracellular
Ca2+ to 1.0 mM caused the plateau level of
[Ca2+]i to drop from 332 ± 26
to 243 ± 27 nM, no alteration in the level of
Ins(1,4,5)P3 production was observed
(Fig. 6). However, further
stepwise decreases in extracellular Ca2+ to 0.3
mM and nominally Ca2+-free resulted in a
decline in NA-mediated Ins(1,4,5)P3 production toward prestimulated
levels. The exact point at which decreases in
[Ca2+]i began to affect
Ins(1,4,5)P3 levels varied between 70 and 170 nM
depending on the cell examined (mean 120 ± 7 nM, 21 cells).
On the return of 1.3 mM Ca2+ to the perfusing
buffer, Ins(1,4,5)P3 levels increased with rising
Ca2+ (Fig.
6). However, again Ins(1,4,5)P3 production was not
sensitive to the full dynamic range of intracellular
Ca2+. Thus, depending on the individual cell,
Ins(1,4,5)P3 levels increased with
[Ca2+]i up to 83260
nM (mean 184 ± 15 nM, 18 cells). After this
point, further increases in intracellular Ca2+ did not
additionally enhance NA-mediated Ins(1,4,5)P3 production.
Stimulation of PLC activity by MCH (10-5 M)
was similarly sensitive to changes in
[Ca2+]i (data not shown).

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FIG. 6. Sensitivity of 1B receptor-mediated
Ins(1,4,5)P3 production to
[Ca2+]i
CHO 1B/M3 cells were transfected with
eGFP-PHPLC 1 and maintained for 48 h
before being loaded with fura-2AM for 1 h and imaged on an epifluorescence
microscope. Trace demonstrates simultaneous measurements for
[Ca2+]i (dotted line) and
Ins(1,4,5)P3 (solid line) in response to
10-4 M NA. Normal KHB containing 1.3
mM Ca2+ was replaced, using the perfusion
system, with KHB containing 1.0, 0.3 mM, or nominally
Ca2+ free (0 Ca2+), as indicated
by the segments, before being returned to normal KHB. NA was present
throughout these manipulations. Note the plateau phase of
Ins(1,4,5)P3 production was insensitive to changes in
[Ca2+]i caused by perfusion with 1.0
mM Ca2+ but declined during perfusion with
0.3 and 0 mM extracellular Ca2+. Trace is
representative of 21 cells.
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Although the above data strongly suggests that Ca2+
feedback could be the driving force for NA-induced Ins(1,4,5)P3
oscillations, the observation that both Ca2+ and
Ins(1,4,5)P3 oscillations could be inhibited by staurosporine
(Fig. 4C) also
suggested a role for protein kinases. As PKC-mediated negative feedback
dynamically controls mGluR5a oscillations
(9,
10,
12), a potential role for PKC
was also investigated. In addition to the effect on NA-mediated oscillatory
responses (Fig. 4C),
staurosporine (10-6 M) also increased levels
of Ins(1,4,5)P3 production in response to maximal concentrations of
NA (10-4 M, 35 cells)
(Fig. 7A). In
contrast, MCH-induced Ins(1,4,5)P3 production in response to
10-6 and 10-8 M MCH
appeared insensitive to staurosporine (Fig.
7, B and C, 34 and 40 cells, respectively).
As staurosporine is a relatively broad spectrum inhibitor of protein
kinases, the role of PKC in NA-induced Ins(1,4,5)P3 oscillations
was further examined by pretreating CHO
1B/M3
cells for 2024 h with the phorbol ester PDBu (1 µM), as
part of an established protocol to down-regulate PKC isoforms
(6). This treatment enhanced
Ins(1,4,5)P3 production in response to both low and high
concentrations of NA (Fig. 8, A
and C). Thus the mean peak increase in
Ins(1,4,5)P3 production in response to 10-7
M NA in PDBu-treated cells was 1.16 ± 0.17 RFU, followed by
a raised plateau level of 1.05 ± 0.17 RFU (29 cells). This represents a
5-fold increase in Ins(1,4,5)P3 production compared with
non-PDBu-treated cells (see above) (Fig. 8,
A and B). Due to this enhanced stimulus
strength, no oscillations in Ins(1,4,5)P3 were observed with
applications of 10-7 M NA.
Ins(1,4,5)P3 production in response to 10-4
M NA was similarly affected. In this case, after PDBu treatment,
the mean peak increase in Ins(1,4,5)P3 production was 3.37 ±
0.14 RFU, whereas the plateau level was 2.96 ± 0.12 RFU, again
representing a 5-fold increase compared with non-treated cells (75 cells)
(Fig. 8, C and
D). In comparison, experiments examining
Ca2+ signals in PDBu pretreated cells loaded with
fura-2AM indicated that under these PKC down-regulated conditions,
10-7 M NA produced a peak and plateau
Ca2+ response, with oscillations in
[Ca2+]i only being observed at lower
agonist concentrations (data not shown). Furthermore, when
Ins(1,4,5)P3 responses in PDBu pretreated
CHO
1B/M3 cells were examined at these lower
concentrations, it was found that concentrations of NA that were sub-threshold
for Ins(1,4,5)P3 production in control conditions (3 x
10-9 to 10-8 M) now
produced oscillations in Ins(1,4,5)P3 (observed in 8 cells)
(Fig. 8E). This
suggests that PKC inhibition alters the stimulus strength of
1B-mediated responses rather than the oscillatory dynamics
of Ins(1,4,5)P3 production. Furthermore, although M3
receptor activation was capable of stimulating oscillations in
Ins(1,4,5)P3 (Fig.
3D), MCH-induced Ins(1,4,5)P3 responses
appeared insensitive to PDBu pretreatment. Thus 10-6
M MCH produced peak and plateau increases in
Ins(1,4,5)P3 of 3.98 ± 0.02 and 2.50 ± 0.16 RFU,
respectively, under control conditions (50 cells), and peak and plateau
increases of 3.51 ± 0.20 and 2.99 ± 0.17 RFU, respectively, in
cells pretreated with 10-6 M PDBu for 24 h
(48 cells).
Differential Ins(1,4,5)P3 Oscillations
Induced by Noradrenergic
1B and mGlu5a
ReceptorsTo compare the Ca2+
feedback-mediated Ins(1,4,5)P3 oscillations observed with
1B receptor activation with the PKC feedback-mediated
mGluR5a Ins(1,4,5)P3 oscillations described previously
(10,
12), the mGluR5a was
transiently transfected into CHO
1B/M3 cells.
Stimulation of mGluR5a-transfected CHO
1B/M3 cells
with L-Glu appeared to stimulate two distinct types of
Ins(1,4,5)P3 oscillation (Fig.
9). Low concentrations of L-Glu (3 x
10-6 M) produced small transient oscillations
in Ins(1,4,5)P3 production of relatively constant magnitude. The
amount of Ins(1,4,5)P3 produced by each transient was similar to
that observed with low levels of NA stimulation (Figs.
3 and
4) and consisted of a 0.18
± 0.02 RFU transient initial peak, followed by further transient
increases of 0.17 ± 0.01 RFU (70 oscillations from 22 to 25 cells)
(Fig. 9). These transient
increases occurred with a frequency of 0.024 ± 0.002 Hz, or one every
41.7 s (24). However, and in
contrast to the effect of increasing levels of
1B receptor
stimulation, oscillations in Ins(1,4,5)P3 production were still
maintained at higher levels of mGluR5a activation. Thus, stimulation with
10-4 M L-Glu produced an oscillatory
Ins(1,4,5)P3 response that was greater in magnitude (1.15 ±
0.18 RFU initial increase, 0.33 ± 0.05 RFU increases for subsequent
oscillations; 66 oscillations from 22 cells) and had a higher frequency 0.044
± 0.003 Hz (1 every 22.7s) than those observed with low concentrations
of L-Glu (Fig. 9).
It should be noted that the levels of Ins(1,4,5)P3 produced by
10-4 M L-Glu in these cells are, in the case
of the initial peak response, markedly greater than that produced by
10-4 M NA
(Fig. 9B). Subsequent
transient increases in Ins(1,4,5)P3 induced by
10-4 M L-Glu are similar in magnitude to the
sustained level produced by 10-4 M NA. In the
case of NA, this amount of Ins(1,4,5)P3 was clearly sufficient to
saturate the Ca2+ release machinery in these cells, and
hence a steady-state level of Ca2+ mobilization is
achieved (Fig. 1A).
The ability of mGluR5a to produce oscillations in Ins(1,4,5)P3 via
a dynamic uncoupling mechanism
(10,
12) must therefore be
essential to enable these large, potentially saturating, increases in
Ins(1,4,5)P3 to elicit a frequency-encoded
Ca2+ response.

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|
FIG. 9. Transient expression of mGluR5a in CHO 1B/M3
cells. A, CHO 1B/M3 cells were
co-transfected with mGluR5a and
eGFP-PHPLC 1 for 48 h before being
imaged on the confocal microscope. Medium was replaced after 24 h to reduce
glutamate accumulation. Cells were perfused with 3 x
10-6 M L-Glu (dotted line) for 210
s, washed for 60 s, and then perfused with 10-4 M
L-Glu (solid line). Trace is representative of 2225
cells. B, mean ± S.E. data for NA- and mGluR5a-induced single
cell Ins(1,4,5)P3 production as detected using
eGFP-PHPLC 1. Data represents the mean
peak data for the initial peak Ins(1,4,5)P3 response (filled
bars), and the average peak height during periods of base-line
Ins(1,4,5)P3 oscillations (secondary; unfilled
bars). Data for 10-4 M NA represent the
initial peak response, followed by the steady-state plateau level of
Ins(1,4,5)P3 production. Numbers of cells are indicated in the
text.
|
|
 |
DISCUSSION
|
---|
Ca2+ oscillations are a ubiquitous mechanism by which
many extracellular signals are encoded in the intracellular environment
(3,
5,
18). These signals control
many cellular processes via Ca2+-sensitive proteins such
as PKC (19) and
calmodulin-dependent protein kinases
(1,
20), which are themselves
regulated by variations in the magnitude, frequency, and spatial distribution
of the Ca2+ signal. The mechanisms responsible for
generating intracellular Ca2+ oscillations have been
subject to considerable debate over the past decade
(5,
6,
7,
8,
21) and center on the key
issue of whether the intracellular messenger of Ca2+
release, Ins(1,4,5)P3, is required to oscillate. Thus,
Ca2+ oscillations may be generated by a steady-state
increase in Ins(1,4,5)P3 via a regenerative CICR process, or
Ca2+ oscillations may follow cyclical changes in
Ins(1,4,5)P3 which are entrained by a negative feedback loop
targeting Ins(1,4,5)P3 production. Recent experiments have begun to
shed some light on the specific mechanisms underlying this important signaling
phenomenon (22). For example,
feedback regulation of Ins(1,4,5)P3 production may occur via RGS
proteins (11), with the
receptor·G-protein·RGS complex regulating
Ins(1,4,5)P3 oscillations and hence the resulting
Ca2+ signal. Crucially, direct single cell measurements
of Ins(1,4,5)P3 production with
eGFP-PHPLC
1 as a biosensor have
emphasized a role for oscillations in Ins(1,4,5)P3 production. By
using this construct, coincident oscillations in Ins(1,4,5)P3 and
Ca2+ have been observed in ATP-stimulated canine kidney
epithelial cells (14).
Furthermore, our own work has demonstrated that oscillations in
Ins(1,4,5)P3 appear to be crucial for mGluR5a-induced
Ca2+ oscillations in CHO cells in which the mGlu5a
receptor is under the control of an inducible expression system
(10,
12). In this case it appears
that the sensitivity of mGluR5a to phosphorylation, and hence inhibition, by
PKC (9) is a key element to the
negative feedback pathway.
Here, we have extended these studies to other PLC-linked GPCRs, and we have
observed an oscillatory Ins(1,4,5)P3 response to stimulation of
1B receptors with low concentrations of NA. Despite the
small magnitude of Ins(1,4,5)P3 response, simultaneous measurements
of Ca2+ signals demonstrated that
Ins(1,4,5)P3 and Ca2+ oscillate
synchronously. Further investigation of the NA-induced Ins(1,4,5)P3
signal indicated that responses were regulated by changes in
[Ca2+]i. Intracellular
Ca2+ mobilization was important, as
Ins(1,4,5)P3 oscillations were blocked by treating cells with
thapsigargin. Furthermore, thapsigargin pretreatment and removal of
extracellular Ca2+ lowered NA-induced
Ins(1,4,5)P3 production relative to a control response in 1.3
mM extracellular Ca2+, suggesting a positive
feedback role for Ca2+ on receptor-mediated Ins
(1,4,5)P3 production. Dual imaging experiments indicated that
receptor-stimulated Ins(1,4,5)P3 production was not sensitive to
the full dynamic range of changes in
[Ca2+]i observed in individual
cells. Thus NA-mediated Ins(1,4,5)P3 production appeared to be
enhanced only by changes in [Ca2+]i
between basal and
200 nM. This limited
Ca2+ sensitivity suggests that only transient base-line
Ca2+ oscillations may be capable of producing an
oscillatory Ins(1,4,5)P3 response. Sinusoidal
Ca2+ oscillations, as they occur on a raised plateau
level of intracellular Ca2+, may be beyond the
Ca2+-sensitive range and hence be unable to stimulate
Ins(1,4,5)P3 oscillations.
PDBu-mediated down-regulation of PKC was shown to enhance NA-mediated
Ins(1,4,5)P3 production in CHO
1B/M3
cells. Importantly, both Ins(1,4,5)P3 and
Ca2+ oscillations were still observed in PKC
down-regulated cells, albeit at concentrations of NA that were sub-threshold
in untreated cells. Thus PKC-mediated feedback inhibition did not appear
essential for the oscillatory responses observed. Furthermore, although
PKC-mediated phosphorylation of
1B receptors has been
demonstrated using phorbol esters to activate PKC, it is notable that these
sites differ from those phosphorylated in response to agonist stimulation of
the receptor (23). In
addition, biochemical studies suggest that G-protein-coupled receptor kinases,
and not PKC, are responsible for homologous phosphorylation and
desensitization of the
1B receptor
(23,
24,
25). It would therefore appear
unlikely that NA-induced Ins(1,4,5)P3 oscillations occur via a
dynamic receptor phosphorylation-uncoupling mechanism mediated by PKC.
Although this differentiates the response from those observed with activation
of mGluR5a (10,
12), it should be noted that
PKC-mediated phosphorylation reactions clearly alter NA-induced
Ins(1,4,5)P3 production (see Figs.
4,
7, and
8). Although this may reflect
an action of PKC on other components of the Ins(1,4,5)P3 signaling
cascade (23), it may also
reflect an alteration in the basal phosphorylation state of the
1B receptor. Thus, treatment of rat-1 cells with
staurosporine or Ro 31-8220 has been shown to reduce basal phosphorylation of
recombinant
1B receptors
(26). The addition of
staurosporine, or pretreatment with PDBu, in this study may therefore enhance
receptor signaling by reducing basal rather than agonist-mediated
phosphorylation of the
1B receptor. Indeed it is interesting
to speculate that the poor efficacy of the
1B receptor may
in part reflect the high level of basal phosphorylation observed with this
receptor. Therefore, PKC phosphorylation may be involved in setting the
sensitivity of the
1B receptor to NA, rather than
dynamically uncoupling the receptor during periods of activation. Furthermore,
despite evidence to the contrary obtained in mouse lacrimal acinar cells
(8), it would appear that the
muscarinic M3 receptor recombinantly expressed in CHO cells is a
poor substrate for PKC, as indicated by the minimal effect of staurosporine
and PBDu pretreatment. The observation that the M3 receptor, which
was unaffected by PKC down-regulation, was also capable of producing
Ins(1,4,5)P3 oscillations further supports a role for oscillations
independent of PKC-mediated dynamic receptor uncoupling.
The dependence of PLC activity on Ca2+ has been
recognized for a considerable length of time
(27,
28,
29,
30,
31), although it is not clear
whether this actually represents a regulatory process in situ. Where
tested, agonist-dependent PLC activity appears to be maximally stimulated by
around 100200 nM
[Ca2+]i
(28,
32,
33), which is comparable with
the results presented in this current study. The results presented here
suggest that for
1B and M3 receptor activation in
CHO cells, changes in intracellular Ca2+ regulate
agonist-mediated responses and produce Ins(1,4,5)P3 oscillations.
Whether the feedback effect of Ca2+ reflects enhancement
of G
q-bound PLC-
isoforms or direct
Ca2+ activation of PLC-
is not clear. Direct
activation of PLC-
may be the mechanism by which
Ca2+ entry, in the absence of activated G-proteins,
stimulated Ins(1,4,5)P3 production in Purkinje cells
(34). However,
PLC-
1 activity appears sensitive to a greater range of
Ca2+ concentration (0.110 µM in
permeabilized HL-60 cells)
(35) than PLC-
. Hence,
the limited sensitivity of PLC activity observed in this current study appears
more in keeping with an effect on PLC-
isoforms. It is of interest to
note from Fig. 5, A and
B, that the secondary, plateau phase of
Ins(1,4,5)P3 production appears particularly sensitive to
regulation by [Ca2+]i. Whether this
indicates recruitment of a Ca2+-sensitive PLC, not
present during initial periods of agonist stimulation, is as yet unclear.
The question therefore arises as to the role of Ca2+
feedback-mediated Ins(1,4,5)P3 oscillations in the highly regulated
Ca2+-release process. Activation of InsP3Rs
is under the dual control of Ins(1,4,5)P3 and
Ca2+ (reviewed in Refs.
36 and
37), and the temporal nature
of these interactions is highly important. Thus, Ins(1,4,5)P3
binding uncovers the stimulatory Ca2+-binding site of
InsP3Rs, allowing Ca2+ to facilitate further
Ca2+ mobilization. In contrast,
Ca2+ binding to InsP3Rs in the absence of
bound Ins(1,4,5)P3 inhibits the receptor
(38). Therefore, increasing
the levels of Ins(1,4,5)P3, either via positive feedback onto PLC
or indeed via dynamic receptor regulation
(10,
12), may partially overcome
this "lateral inhibition" of InsP3Rs and hence further
increase Ca2+ release. It should be noted that this does
not represent an uncontrolled positive feedback loop onto PLC activation. The
limited sensitivity of PLC activity to Ca2+ implies that
further Ca2+ increases will still cause lateral
inhibition and hence prevent Ins(1,4,5)P3 from releasing
Ca2+. The intrinsic inactivation of the
InsP3R and dissociation of Ins(1,4,5)P3
(39) reduces
[Ca2+]i and hence
Ins(1,4,5)P3 production. The delay between
Ca2+ oscillations, which is controlled by the strength
of agonist stimulus, is likely to reflect the amount of
Ins(1,4,5)P3 being produced in combination with some intrinsic
property of the regenerative CICR process such as intracellular store
refilling.
In summary, Ca2+ oscillations in CHO cells are
capable of stimulating transient increases in Ins(1,4,5)P3 via a
positive feedback effect on PLC. Due to the demonstrated sensitivity of PLC
activity to Ca2+, only Ca2+
increases of a transient baseline spiking nature would be expected to cause
positive feedback. Hence sinusoidal Ca2+ oscillations
may occur in the absence of any oscillatory Ins(1,4,5)P3 signal
(12). Although
Ca2+ feedback-mediated Ins(1,4,5)P3
oscillations appear smaller than those caused by dynamic receptor uncoupling,
this work demonstrates that both positive and negative feedback pathways can
lead to the same result, namely an oscillatory Ins(1,4,5)P3
response. Positive feedback onto PLC is likely to occur in all situations
where Ca2+ is oscillating in the appropriate range, and
there is sufficient G
q-mediated activation of PLC. In
contrast, PKC-mediated dynamic receptor uncoupling is likely to occur only
where the receptor is a suitable substrate for PKC.
 |
FOOTNOTES
|
---|
* This work was supported by the Wellcome Trust Grant 062495. The costs of
publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed. Tel.: 0116 252 5249; Fax: 0116 252
5045; E-mail:
KWY1{at}LE.AC.UK.
1 The abbreviations used are: GPCR, G-protein coupled receptor; CICR,
Ca2+ induced Ca2+ release;
eGFP-PHPLC
1, PH domain of
PLC
1 tagged with enhanced green fluorescent protein;
Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; InsP3R,
inositol 1,4,5-trisphosphate receptor; MCH, methacholine; NA, noradrenaline;
PKC, protein kinase C; PDBu, phorbol dibutyrate; PLC, phospholipase C; RFU,
relative fluorescent units (F/F0); PH,
pleckstrin homology; CHO, Chinese hamster ovary; KHB, Krebs-Henseleit buffer;
RGS, regulators of G-protein signaling. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. E. Hermans (Laboratoire de Pharmacologie, Université
Catholique de Louvain, Brussels) for creating the CHO-lac-mGlu5a cell line and
Debbie Channing for initial characterization of
CHO
1B/M3 cells.
 |
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