Role of Ca2+ Feedback on Single Cell Inositol 1,4,5-Trisphosphate Oscillations Mediated by G-protein-coupled Receptors*

Kenneth W. Young {ddagger}, 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.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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) {delta}1 tagged with enhanced green fluorescent protein (eGFP-PHPLC{delta}1). Activation of noradrenergic {alpha}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 {alpha}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 {alpha}1B receptors, produced a sustained increase in intracellular Ca2+ even at low levels of agonist stimulation. Confocal imaging of eGFP-PHPLC{delta}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 {alpha}1B receptor-mediated Ins(1,4,5)P3 oscillations result from a positive feedback effect of Ca2+ onto phospholipase C. The mechanisms underlying {alpha}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, 381–382). For {alpha}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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{delta}1 tagged with enhanced green fluorescent protein (eGFP-PHPLC{delta}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 {alpha}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 {alpha}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Plasmid Transfection—CHO cells stably expressing noradrenergic {alpha}1B (2.3 pmol/mg protein) and muscarinic M3 receptors (3.4 pmol/mg protein) (CHO{alpha}1B/M3) were maintained in minimum Eagle's medium-{alpha} (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-{beta}-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{alpha}1B/M3 cells, 0.5 µg of mGluR5a in pcDNA3 (Invitrogen) was co-transfected with 0.5 µg of eGFP-PHPLC{delta}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{delta}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 1.5–2.5 Hz. Increases in cellular Ins(1,4,5)P3 were detected by measuring the translocation of eGFP-PHPLC{delta}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{alpha}1B/M3 cells with eGFP-PHPLC{delta}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{delta}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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Receptor-dependent Ca2+ Oscillations in CHO Cells—The temporal patterns of intracellular Ca2+ signals in CHO cells co-expressing noradrenergic {alpha}1B and muscarinic M3 receptors were compared with those elicited by the metabotropic glutamate receptor mGluR5a. The {alpha}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{alpha}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{alpha}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."

 

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{delta}1) and Ca2+ mobilization (using fura-2) demonstrated that although the {alpha}1B and M3 receptors are co-expressed at similar levels in the CHO{alpha}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{delta}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{alpha}1B/M3 cells. Cells were transfected with eGFP-PHPLC{delta}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.

 

Confocal imaging more clearly revealed the presence of an oscillatory Ins(1,4,5)P3 response to low concentrations of NA in CHO{alpha}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, 80–86 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{alpha}1B/M3 cells. CHO{alpha}1B/M3 cells were transfected with eGFP-PHPLC{delta}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.

 

Role of Ca2+ and PKC in Single Cell Ins(1,4,5)P3 Oscillations—NA-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{alpha}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{alpha}1B/M3 cells were transfected with eGFP-PHPLC{delta}1 and imaged on the confocal microscope after 48 h. Right panels, in a separate set of experiments, CHO{alpha}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.

 

To investigate further the role of [Ca2+]i in NA-mediated IP3 production, CHO{alpha}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 {alpha}1B receptor-mediated Ins(1,4,5)P3 production. A, eGFP-PHPLC{delta}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).

 

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 83–260 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 {alpha}1B receptor-mediated Ins(1,4,5)P3 production to [Ca2+]i CHO{alpha}1B/M3 cells were transfected with eGFP-PHPLC{delta}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.

 

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).



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FIG. 7.
Effect of staurosporine on receptor-mediated Ins(1,4,5)P3 production. CHO{alpha}1B/M3 cells were transfected with eGFP-PHPLC{delta}1 and maintained for 48 h before being imaged on the confocal microscope. A, 10-4 M NA was applied to the cells for 200 s before the addition of 10-6 M staurosporine (stauro) in the continued presence of NA (indicated by the dotted line). B, and C, experiments conducted as described in A except cells were stimulated with 10-6 and 10-8 M MCH, respectively. Data are representative of 35 cells for A, 40 cells for B, and 34 cells for C.

 

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{alpha}1B/M3 cells for 20–24 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{alpha}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 {alpha}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).



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FIG. 8.
Effect of PKC down-regulation on receptor-mediated Ins(1,4,5)P3 production. CHO{alpha}1B/M3 cells were transfected with eGFP-PHPLC{delta}1 for 24 h before being treated for a further 24 h with 10-6 M PDBu to down-regulate PKC isoforms. Receptor-mediated Ins(1,4,5)P3 production was then imaged on the confocal microscope. A and B, cells were perfused with 10-7 M NA as indicated by the dotted line in control cells (B), or cells pretreated with PDBu (A). C and D as A except cells were perfused with 10-4 M NA. E, cells pretreated with PDBu for 24 h were perfused with 5 x 10-9 M NA. Traces are representative examples from 29 cells in A, 75 cells in C, and 8 oscillating cells in E.

 

Differential Ins(1,4,5)P3 Oscillations Induced by Noradrenergic {alpha}1B and mGlu5a Receptors—To compare the Ca2+ feedback-mediated Ins(1,4,5)P3 oscillations observed with {alpha}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{alpha}1B/M3 cells. Stimulation of mGluR5a-transfected CHO{alpha}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 {alpha}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{alpha}1B/M3 cells. A, CHO{alpha}1B/M3 cells were co-transfected with mGluR5a and eGFP-PHPLC{delta}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 22–25 cells. B, mean ± S.E. data for NA- and mGluR5a-induced single cell Ins(1,4,5)P3 production as detected using eGFP-PHPLC{delta}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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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{delta}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 {alpha}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{alpha}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 {alpha}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 {alpha}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 {alpha}1B receptor. Thus, treatment of rat-1 cells with staurosporine or Ro 31-8220 has been shown to reduce basal phosphorylation of recombinant {alpha}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 {alpha}1B receptor. Indeed it is interesting to speculate that the poor efficacy of the {alpha}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 {alpha}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 100–200 nM [Ca2+]i (28, 32, 33), which is comparable with the results presented in this current study. The results presented here suggest that for {alpha}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{alpha}q-bound PLC-{beta} isoforms or direct Ca2+ activation of PLC-{delta} is not clear. Direct activation of PLC-{delta} 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-{delta}1 activity appears sensitive to a greater range of Ca2+ concentration (0.1–10 µM in permeabilized HL-60 cells) (35) than PLC-{beta}. Hence, the limited sensitivity of PLC activity observed in this current study appears more in keeping with an effect on PLC-{beta} 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{alpha}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. Back

{ddagger} 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{delta}1, PH domain of PLC{delta}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. Back


    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{alpha}1B/M3 cells.



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