Dual Regulation of Calcium Mobilization by Inositol 1,4,5-Trisphosphate in a Living Cell

Svetlana Tertyshnikovaa and Alan Feina
a From the Department of Physiology, University of Connecticut Health Center, Farmington, Connecticut 06030-3505

Correspondence to: Alan Fein, Dept. of Physiology, University of Connecticut Health Center, Farmington CT, 06030-3505. Fax:860-679-1269 E-mail:afein{at}neuron.uchc.edu.


  Abstract
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

Changes in cytosolic free calcium ([Ca2+]i) often take the form of a sustained response or repetitive oscillations. The frequency and amplitude of [Ca2+]i oscillations are essential for the selective stimulation of gene expression and for enzyme activation. However, the mechanism that determines whether [Ca2+]i oscillates at a particular frequency or becomes a sustained response is poorly understood. We find that [Ca2+]i oscillations in rat megakaryocytes, as in other cells, results from a Ca2+-dependent inhibition of inositol 1,4,5-trisphosphate (IP3)–induced Ca2+ release. Moreover, we find that this inhibition becomes progressively less effective with higher IP3 concentrations. We suggest that disinhibition, by increasing IP3 concentration, of Ca2+-dependent inhibition is a common mechanism for the regulation of [Ca2+]i oscillations in cells containing IP3-sensitive Ca2+ stores.

Key Words: megakaryocyte, protein kinase C, pleckstrin, IP3-5-phosphatase, platelets


  INTRODUCTION
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

Calcium is a universal intracellular signaling agent involved in a myriad of processes from fertilization to cell death (Berridge et al. 1998 ). Changes in cytosolic free calcium ([Ca2+]i) are well documented for cells stimulated by many hormone and growth factor agonists that generate the second messenger inositol 1,4,5-trisphosphate (IP3).1 [Ca2+]i signals can be a single transient or a sustained increase, but very often take the form of repetitive spikes or oscillations. The frequency and amplitude of [Ca2+]i oscillations are essential for initiating numerous cellular processes, including selective stimulation of gene expression (Dolmetsch et al. 1998 ; Li et al. 1998 ) and the activation of specific enzymes (De Koninck and Schulman 1998 ). It has been observed that as the concentration of agonist is increased [Ca2+]i oscillations increase in frequency, eventually becoming a sustained [Ca2+]i elevation (Jacob et al. 1988 ; Wakui et al. 1989 ; Heemskerk et al. 1993 ). A similar phenomenon has also been seen when cells are dialyzed with increasing concentrations of the nonmetabolized IP3 analogue, inositol 1,4,5 trisphosphorothioate (Petersen et al. 1991 ). However, the mechanism by which the [Ca2+]i oscillation frequency increases and how the response changes into a sustained [Ca2+]i elevation is not understood.

Many models of agonist-induced [Ca2+]i oscillations in nonexcitable cells require some form of Ca2+-dependent inhibition of IP3-induced Ca2+ release as a fundamental component (Fewtrell 1993 ). In these models, released Ca2+ feeds back to inhibit further release of Ca2+ by IP3 (Payne et al. 1988 ; Ogden et al. 1990 ; Ilyin and Parker 1994 ; Oancea and Meyer 1996 ; Carter and Ogden 1997 ). However, it is not clear how these models could explain the increase in the [Ca2+]i oscillation frequency with increased agonist concentration described above. Or for that matter how the [Ca2+]i oscillation changes into a sustained [Ca2+]i elevation. A possible answer might come from in vitro studies, which have shown that the extent of Ca2+-dependent inhibition may be regulated by the concentration IP3. For example, the inhibition by Ca2+ of IP3-induced Ca2+ release from cerebellar microsomes (Joseph et al. 1989 ; Combettes et al. 1994 ; Hannaert-Merah et al. 1995 ) and permeabilized A7r5 smooth muscle cells (Bootman et al. 1995 ) decreases as the IP3 concentration is elevated. Likewise, a similar effect is seen at the level of the single IP3-gated Ca2+ channel (Kaftan et al. 1997 ; Mak et al. 1998 ). Whether or not this decrease of Ca2+-dependent inhibition as the IP3 concentration is elevated occurs in intact cells is not known. The experiments described herein were designed to extend these in vitro findings to an intact cell, the rat megakaryocyte. We show for the first time, in an intact cell, that Ca2+-dependent inhibition of IP3-induced Ca2+ release becomes progressively less effective with higher IP3 concentrations.


  METHODS
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

The methods used in these experiments have been fully described in previous publications (Tertyshnikova and Fein 1997 , Tertyshnikova and Fein 1998 ; Tertyshnikova et al. 1998 ; Lu et al. 1999 ). They are described briefly below.

Rat Megakaryocytes
Bone marrow is obtained from the tibial and femoral bones of adult Wistar rats. After filtration through a 75-µm nylon mesh to eliminate large masses of cells, the bone marrow suspension is spun and washed twice before incubation in standard external solution containing (mM): 140 NaCl, 5 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, 10 HEPES, pH 7.4, supplemented by 0.1% BSA. Megakaryocytes are clearly distinguished from other bone marrow cells on the basis of their large size (25–50 µm) and multilobular nucleus (Uneyama et al. 1993 ; Kapural and Fein 1997 ). All experiments are done within 2–6 h after preparation at room temperature (2325°C).

measurement of [Ca2+]i and Photolysis of Caged Compounds
Megakaryocytes are viewed through a coverslip forming the bottom of the recording chamber using a Diaphot microscope equipped with a Fluor 100x 1.3 NA oil immersion lens (Nikon Inc.). Single cell fluorometry is accomplished using an Ionoptix photon-counting fluorescence subsystem with a dual excitation light source (designed by Dr. D. Tillotson; Ionoptix) using Oregon Green 488 BAPTA-1 (OGB488) as the [Ca2+]i indicator. fluorescence intensity is measured on-line using the Ionwizard program (IonOptix). For photolysis of caged compounds, pulses of ultra violet light (290–370 nm) are applied to the cell through the second channel of the dual excitation light source. Calibration of photolysis in the microscope was by measurement of the fluorescence change produced in the pH dye 2',7'-bis(carboxy-ethyl)-5(6)-carboxyfluorescein by protons released during the photolysis of NPE-caged ATP (Fink et al. 1999 ). A 50-s flash uncaged ~50% of the caged ATP.

Chemicals
The "cell-permeant" AM ester and the "cell-impermeable" hexapotassium salt of OGB488 are obtained from molecular Probes, Inc. Caged IP3 and caged GPIP2 [1-(alpha-glycerophosphoryl)-myo-inositol 4,5-diphosphate, P4(5)-1-(2-nitrophenyl) ethyl ester] are from Calbiochem Corp. GF109203X is from Biomol and 2,3-diphosphoglycerate (2,3-DPG) is from Sigma Chemical Co.

Cell Loading of Caged Compounds
The cell-permeant AM ester of OGB488 is dissolved in DMSO and stored at -20°C. For the experiments not using patch clamping, cells are transferred onto glass coverslips and incubated with 2.5–5 µM OGB488/AM for 30 min. For the experiment with caged calcium, the cells are first incubated with 10–30 µM caged calcium for at least 2 h. The final concentration of DMSO is always <0.1%. The coverslips with adherent cells are then washed several times with the standard external solution, and kept in the dark until use. For the other experiments, caged IP3 or caged GPIP2 together with OGB488 hexapotassium salt are included in the intrapipette solution at 100 and 200 µM, respectively [composition (mM): 20 KCl, 120 K-glutamate, 1 MgCl, 2 Na-GTP, 10 HEPES, pH 7.3]. Standard whole-cell patch-clamp recording techniques are used to voltage clamp and internally dialyze single megakaryocytes. Membrane current is monitored using an Axopatch-1D patch clamp amplifier (Axon Instruments). For most cells, 5–6 min is required for the OGB488 fluorescence signal to equilibrate in the patch-clamped cell.

Agonist Application
ADP or the mixture of ADP with GF109203X are dissolved in the standard external solution and applied directly to single megakaryocytes using a DAD-6 computer-controlled local superfusion system (ALA Scientific Instruments, Inc.). The output tube of the micromanifold (100 µm inside diameter) is placed within ~200 µm of the cell and the puff pressure is adjusted to achieve rapid agonist application while avoiding any mechanical disturbance of the cell.


  RESULTS
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

To study Ca2+-dependent inhibition of IP3-induced Ca2+ release, we performed paired-pulse experiments in rat megakaryocytes that are a convenient model for studying Ca2+ signaling in nonexcitable cells, because they express only an IP3-sensitive Ca2+ store and lack a ryanodine-sensitive Ca2+ store (Uneyama et al. 1993 ). For the first pulse of IP3, in a paired-pulse experiment, the intracellular increase of IP3, resulting from photorelease from caged IP3, causes a transient release of Ca2+ lasting a few hundred milliseconds. After the response to the first pulse, there is a period of desensitization lasting several seconds, during which responses to a second pulse of IP3 are diminished in amplitude (see Fig 2 A). The available evidence indicates that this period of desensitization is due to Ca2+-dependent inhibition of IP3-induced Ca2+ release (Ogden et al. 1990 ; Payne et al. 1988 , Payne et al. 1990 ; Ilyin and Parker 1994 ; Oancea and Meyer 1996 ; Carter and Ogden 1997 ).



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Figure 1. Dose–response curve for IP3-mediated Ca2+ release. (A) IP3-mediated [Ca2+]i response for UV flashes of increasing duration. Caged IP3 (100 µM) and OGB488 (200 µM) were included in the patch pipette solution. (B) Normalized peak amplitude (R/Rmax) of the IP3-mediated [Ca2+]i response for the data in A as a function of the flash duration, where Rmax is the maximum peak amplitude observed. (C) Normalized peak amplitude (R/Rmax) of the IP3-mediated [Ca2+]i response for 10 cells as a function of the normalized flash duration. The data in B and C are well fit with the Hill equation R/Rmax = 1/[1 + (d1/2/d)n], with n = 7, where d1/2 is the flash duration that gives a peak response of 1/2 the maximum amplitude. (B) d1/2 = 108 ms, (C) d1/2 = 1.



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Figure 2. Paired pulse experiment to measure recovery from desensitization in rat megakaryocytes loaded with either caged-IP3 or -GPIP2. In each trace of A and B, the rise in [Ca2+]i was monitored by measuring OGB488 fluorescence intensity and is expressed as {Delta}F (counts/millisecond). For each trace in A and B, the cell was stimulated by two flashes of UV light, which released IP3 in A and GPIP2 in B. For the cell in A, 100 µM caged IP3 and 200 µM OGB488 were included in the patch pipette solution. For the cell in B, 100 µM caged GPIP2 and 200 µM OGB488 were included in the patch pipette solution. See METHODS for further experimental details.

In rat basophilic leukemia cells, maximal desensitization of the response to the second pulse of IP3 is observed for a first pulse of IP3 that produced a [Ca2+]i response of near maximal amplitude (Oancea and Meyer 1996 ). Therefore, we began our experiments by first measuring the power dependence of IP3-induced Ca2+ release (Fig 1). In Fig 1B and Fig C, we plot the normalized peak amplitude (R/Rmax) of the IP3-mediated [Ca2+]i response as a function of the flash duration, which is directly proportional to IP3 concentration. As can be seen in Fig 1 C, the data are well fit with the Hill equation with a coefficient of n = 7. For the 10 cells in Fig 1 C, the flash duration that produced a response of half the maximal amplitude was 203 ± 95 ms (mean ± SD).

We found that maximal desensitization was observed when the flash duration in a paired-pulse experiment produced a response just below that which gives a response of saturating amplitude. An example of such an experiment can be seen in Fig 2 A for which, after the release of Ca2+ produced by the photorelease of IP3, there is a period of desensitization during which a subsequent increase in IP3 releases less calcium. As the time interval between the pulses of IP3 increases, the response to the second pulse recovers back to that of the first. The desensitization is not due to emptying of the Ca2+ stores, because desensitization of the second response disappears if the duration of the second flash is increased threefold, thereby saturating the amplitude of the second response (n = 6 cells, data not shown). The desensitization also disappears if the duration of both flashes is increased three- to fourfold, thereby saturating the response amplitude of the response to each flash (n = 3 cells, data not shown). These findings are similar to what was found for rat basophilic leukemia cells (Oancea and Meyer 1996 ), for which it was concluded that a two- to threefold decrease in IP3 sensitivity was sufficient to explain the reduced amplitude of the response to the second pulse of IP3, and we suggest that the same is true for rat megakaryocytes.

The experiments described above establish the basic conditions for measuring the time course of recovery in a paired-pulse experiment. Having established these conditions, we can now turn to the central question of this investigation, whether Ca2+-dependent inhibition of IP3-induced Ca2+ release becomes progressively less effective with higher IP3 concentrations. For this purpose, we used procedures that would increase the lifetime of IP3, by slowing down its hydrolysis. We began by comparing the time course for the recovery from desensitization produced by IP3 injection with the time course for recovery from desensitization produced by injection of a hydrolysis-resistant analogue of IP3, namely GPIP2. GPIP2 is a less potent but fully active analogue of IP3 that is poorly metabolized, and the caged form of GPIP2 has been used to mobilize Ca2+ from IP3-sensitive Ca2+ stores (Berven and Barritt 1994 ). As with IP3, the flash duration when using caged GPIP2 is set to give a response just below that which gives a response of saturating amplitude. After the release of Ca2+ produced by the photorelease of GPIP2 (Fig 2 B), the cell recovers its sensitivity much faster than in Fig 2 A. It would appear from the results in Fig 2 that the recovery from desensitization accelerates when the rate of hydrolysis of IP3 is slowed down. This is the opposite of what one would expect if the recovery from desensitization were following the time course for the hydrolysis of IP3. We assume that the acceleration in the rate of recovery is due to the decreased rate of hydrolysis of GPIP2 compared with IP3. If this assumption is correct, then we should be able to accelerate the recovery from desensitization produced by photoreleased IP3 to a time course similar to that produced by photoreleased GPIP2 by inhibiting the IP3-5-phosphatase, the enzyme which hydrolyses IP3.

Accordingly, in Fig 3, we compare the time course for recovery after photorelease of IP3, in the presence and absence of 2,3-DPG (2,3-diphosphoglycerate), an inhibitor of the IP3-5-phosphatase (Shears 1989 ; Wood et al. 1990 ). In Fig 3, we plot the ratio (A2/A1) as a function of the time interval between the pulses, where A2 is the peak amplitude of the response to the second pulse of IP3 and A1 is the peak amplitude of the response to the first pulse of IP3 (Fig 2). Each group of recovery data in Fig 3 was fit with Equation 1 to obtain an estimate of the average time for recovery for each experimental condition.



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Figure 3. Time course of recovery from desensitization in rat megakaryocytes loaded with either caged IP3 or GPIP2. The data points were obtained from paired-pulse experiments similar to those in Fig 2. Each data point represents the ratio (A2/A1) of the peak amplitude of the response to the second pulse of IP3 (A2) to the peak amplitude of the response to the first pulse of IP3 (A1) as a function of the time interval between the pulses (Fig 2). We used 10 mM 2,3-DPG and 10 µM GF109203X in these experiments. Although a concentration of 10 mM for 2,3-DPG may seem high, it is actually quite reasonable as the Ki for inhibition of IP3-5-phosphatase is in the millimolar range (for example, the Ki = 4 mM in squid photoreceptors; Wood et al. 1990 ). See METHODS for further experimental details and the text for an explanation of the solid curves and the {tau} values associated with them.


(1)

In Equation 1, the 1.5-s time delay is the approximate time to peak for the response to IP3 or GPIP2. For IP3 alone, {tau} = 15 s (n = 8 cells) and for IP3 with 2,3-DPG, {tau} = 4.2 s (n = 6 cells). Also included in Fig 3 are recovery data for GPIP2 that were fit with {tau} = 2.6 s (n = 5 cells) and data for IP3 in the presence of GF109203X, which were fit with {tau} = 5.4 s (n = 11 cells).

GF109203X is a cell-permeable inhibitor of PKC that has been used effectively to inhibit PKC in platelets (Toullec et al. 1991 ). Inhibition of PKC in platelets causes an approximately threefold increase in IP3 levels in thrombin-activated platelets (King and Rittenhouse 1989 ). This is thought to occur via the inhibition of the phosphorylation of pleckstrin, the major substrate of PKC in platelets, because phosphorylated pleckstrin has been shown to activate the IP3-5-phosphatase (Auethavekiat et al. 1997 ). Therefore, inhibition of PKC by GF109203X should inhibit the hydrolysis of IP3 by the 5-phosphatase and consequently prolong the lifetime of IP3. The experimental data in Fig 2 and Fig 3 clearly suggest that the recovery from desensitization is accelerated when the rate of hydrolysis of IP3 is slowed down. This suggests that the extent of Ca2+-dependent inhibition is diminished when the lifetime of IP3 is increased.

To be certain that the findings in Fig 3 are not somehow the result of an effect of GF109203X, 2,3-DPG, or GPIP2 on the power dependence of IP3-induced Ca2+ release, we carried out the experiment presented in Fig 4. The data in Fig 4 clearly show that the power dependence for GPIP2- and IP3-induced Ca2+ release in the presence of GF109203X or 2,3-DPG are no different than the power dependence of IP3-induced Ca2+ release itself. The flash duration that produced a response of half the maximal amplitude was 108 ± 39 ms (n = 8 cells) for GPIP2, 155 ± 54 ms (n = 5 cells) for IP3 in the presence of 2,3-DPG, and 121 ± 44 ms (n = 8 cells) for IP3 in the presence of GF109203X. The flash duration for half-maximal amplitude for GPIP2 and IP3 in the presence of GF109203X are significantly different than that for IP3 at the P = 0.05 level using the unpaired Student's t test. However, the flash duration for half-maximal amplitude for IP3 in the presence of 2,3-DPG is not significantly different than that for IP3. Hence the findings in Fig 3 are consistent with our suggestion that the extent of Ca2+-dependent inhibition is diminished when the lifetime of IP3 is increased.



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Figure 4. Dose–response curve for GPIP2-mediated Ca2+ release and IP3-mediated Ca2+ release in the presence of 2,3-DPG or GF109203X. OGB488 (200 µM) together with either caged IP3 (100 µM) or GPIP2 (100 µM) were included in the patch pipette solution. We used 10 mM 2,3-DPG and 10 µM GF109203X in these experiments. As in Fig 1 C, we plot the normalized peak amplitude (R/Rmax) of the IP3- or GPIP2-mediated [Ca2+]i response as a function of the normalized flash duration, where Rmax is the maximum peak amplitude of the [Ca2+]i response. Also included in this figure is the data from Fig 1 C. The data are well fit with the Hill equation R/Rmax = 1/[1 + (d1/2/d)n], with n = 7 and d1/2 = 1.

Based on the data in Fig 2 and Fig 3, we predict that the falling phase of the response to the uncaging of GPIP2 should be dominated by the inhibitory effect of elevated [Ca2+]i on further Ca2+ release and the removal of Ca2+ from the cytoplasm. That is, the hydrolysis of GPIP2 by the 5-phosphatase should have minimal effect on the falling phase of the response. Accordingly, the falling phase of the response to the uncaging of GPIP2 should be greatly prolonged when compared with that for the uncaging of IP3, especially as the amount of IP3 or GPIP2 uncaged is increased. In Fig 5, we compare the time course of the [Ca2+]i response to the uncaging of IP3 with that for the uncaging of GPIP2. As the duration of the uncaging flash is increased from 150 to 2,000 ms, it can be seen that the falling phase of the response to GPIP2 is greatly prolonged when compared with that for IP3. Results similar to those in Fig 5 were seen in two additional cells each.



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Figure 5. Comparison of the time course of the [Ca2+]i response to the uncaging of IP3 or GPIP2 in rat megakaryocytes. Flashes of 150-, 300-, 1,000-, and 2,000-ms duration were used for uncaging with each cell. Changes in [Ca2+]i were monitored by measuring OGB488 fluorescence intensity and are expressed as {Delta}F (counts/millisecond). Results similar to those shown were seen in two other cells each.

As mentioned above, Ca2+-dependent inhibition of IP3-mediated Ca2+ release is thought to play a central role in the generation of [Ca2+]i oscillations. Also, megakaryocytes exhibit [Ca2+]i oscillations when exposed to ADP (Tertyshnikova and Fein 1997 ; Uneyama et al. 1993 ). To examine how the lessening of Ca2+-dependent inhibition will affect an agonist-induced [Ca2+]i oscillation, we examined the effect of GF109203X on ADP-induced [Ca2+]i oscillations. As shown in Fig 6, in the presence of GF109203X, ADP causes a plateau-like rise in [Ca2+]i (n = 3 cells).The experiment in Fig 6 was carried out in a Ca2+-free external solution in presence of 1 mM BAPTA, to rule out the possibility that the effect of GF109203X was on Ca2+ influx. Results similar to those in Fig 6 were obtained when the experiment was performed in standard external solution that contains 2 mM calcium (n = 4 cells, data not shown). GF109203X is also a less potent inhibitor of cAMP-PK; however, the effect of GF109203X on rat megakaryocytes is entirely different from what we have found when inhibiting cAMP-PK in these cells (Tertyshnikova and Fein 1998 ).



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Figure 6. Effect of the PKC inhibitor, GF109203X, on ADP-induced [Ca2+]i oscillations in a rat megakaryocyte. Changes in [Ca2+]i were monitored by measuring OGB488 fluorescence intensity and are expressed as {Delta}F (counts/millisecond). GF109203X (15 µM) and ADP (100 µM) were applied to the cell via the local superfusion system. The cell was loaded with OGB488 using the cell permeable AM ester of OGB488. Similar results were seen in five other cells. See METHODS for further experimental details.

The results in Fig 6 are very similar to those obtained by examining the effect of another PKC inhibitor, staurosporine, on ATP-induced [Ca2+]i oscillations monitored as a calcium-activated potassium current oscillation (Uneyama et al. 1993 ). These workers (Uneyama et al. 1993 ) speculated that the effect on [Ca2+]i oscillations, of inhibiting PKC with staurosporine, resulted from an inhibition of the Ca2+ pump. To investigate whether GF109203X affects Ca2+ uptake and/or extrusion, we used caged Ca2+ for the experiment in Fig 7. The time course of the fall in [Ca2+]i after the flash-induced rise in [Ca2+]i should reflect the activity of Ca2+ sequestration and/or extrusion mechanisms (see Tertyshnikova and Fein 1998 ; Tertyshnikova et al. 1998 ). As can be seen in Fig 7, photoreleased [Ca2+]i declined at the same rate in the presence and absence of GF109203X. In the experiment shown in Fig 7, cyclopiazonic acid, an inhibitor of the smooth endoplasmic reticulum calcium ATPase in platelets (Papp et al. 1993 ), was used as a positive control for inhibition of Ca2+ sequestration. Similar results as those in Fig 7 were seen in two other cells. The results in Fig 7 appear to convincingly rule out inhibition of the Ca2+ pump as an explanation for the findings in Fig 6.



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Figure 7. The PKC inhibitor GF109203X does not affect the rate of calcium removal from the cytoplasm. The cell was loaded with caged Ca2+ and OGB488 using the cell-permeant forms of each molecule as described in METHODS. [Ca2+]i spikes resulting from photorelease of caged Ca2+ are shown superimposed for the purpose of comparison. GF 109203X had no effect on the time course of the fall in [Ca2+]i after photolysis of caged-Ca2+. Cyclopiazonic acid (CPA) was applied to the cell to serve as a positive control for inhibition of Ca2+ sequestration. GF109203X (30 µM) and CPA (5 µM) were applied to the cell via the local superfusion system as described in METHODS.

Based on the data of Fig 2, Fig 3, and Fig 6, we would expect that in response to multiple injections of IP3, the rise in [Ca2+]i would become plateau-like when the hydrolysis of IP3 is slowed down. Accordingly, in Fig 8, we compare the responses to multiple flashes, which photorelease IP3, in the presence and absence of 2,3-DPG. As can be seen in Fig 8 A, the response to the first flash that photoreleases IP3 is large, and the responses to subsequent flashes are greatly reduced in amplitude. Based on the results presented in Fig 2 and Fig 3, the finding in Fig 8 A is as expected. In contrast, in the experiment of Fig 8 B, in which 10 mM 2,3-DPG was included in the patch pipette to inhibit the IP3-5-phosphatase, a series of flashes that photorelease IP3 produce a sustained elevation of [Ca2+]i. Likewise a series of flashes that photorelease IP3 produce a sustained elevation of [Ca2+]i in the presence of GF109203X (Fig 8 D). Furthermore, a train of flashes that photorelease the hydrolysis-resistant IP3-analogue GPIP2 also produce a sustained elevation of [Ca2+]i (Fig 8 C).



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Figure 8. Elevation of [Ca2+]i resulting from multiple flashes that photorelease caged IP3 or GPIP2. (A) Caged IP3, (B) caged IP3 in the presence of 10 mM 2,3-DPG, (C) caged GPIP2, and (D) caged IP3 in the presence of 5 µM GF109203X. Either caged IP3 (100 µM) and OGB488 (200 µM), or caged GPIP2 (100 µM) and OGB488 (200 µM) were included in the patch-pipette solution. The cell in A was stimulated with 11 flashes spaced 1.8-s apart, the cell in B was stimulated with 6 flashes spaced 3.6-s apart, the cell in C was stimulated with 16 flashes spaced 1.2-s apart, and the cell in D was stimulated with 11 flashes spaced 1.2-s apart. Similar results as those in A–D were seen in at least three to five other cells each.

The simplified diagram in Fig 9 summarizes our findings, emphasizing the dual regulation of calcium mobilization by IP3. For the sake of simplicity, GPIP2 has been left out of the figure. The heavy lines in Fig 9 are meant to represent the release of Ca2+ by IP3 and the disinhibition of Ca2+-dependent inhibition of IP3-mediated Ca2+ release by increasing IP3 concentration. We show this disinhibition as acting via calmodulin because recently published experiments have indicated that Ca2+-dependent inhibition of IP3 -mediated Ca2+ release for the type 1 IP3 receptor (IP3-R) is mediated by calmodulin (Michikawa et al. 1999 ) (see DISCUSSION).



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Figure 9. Simplified diagram illustrating disinhibition of Ca2+-dependent inhibition of IP3-mediated Ca2+ release with higher IP3 concentrations (heavy arrow). ADP is shown as activating phospholipase C (Pl-C) via the GTP-binding protein (Gq) to form diacylglycerol (DAG) and IP3. IP3 is shown as causing the release of Ca2+ (heavy arrow) and DAG is shown as activating PKC, which in turn phosphorylates pleckstrin (pleck). Phosphorylated pleckstrin is shown as activating the IP3-5 phosphatase (IP3ase), which inactivates IP3 by hydrolyzing it to inositol 1,4-bisphosphate (IP2). GF109203X and 2,3DPG are shown as inhibiting PKC and the IP3ase, respectively. Released Ca2+ is shown as feeding back via calmodulin (CaM) to inhibit further release of Ca2+ by the IP3-R.


  DISCUSSION
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

Our results demonstrate for the first time an important property of [Ca2+]i signaling in intact cells: an increase in the lifetime of IP3 brings about a decrease in Ca2+-dependent inhibition. These findings suggest a mechanism by which high concentrations of intracellular IP3 can cause cells to maintain an elevated level of [Ca2+]i. Indeed, this may explain the occurrence of sustained [Ca2+]i elevations at high agonist concentrations (Jacob et al. 1988 ; Wakui et al. 1989 ; Heemskerk et al. 1993 ) and when cells are dialyzed with high concentrations of the nonmetabolized IP3 analogue inositol 1,4,5 trisphosphorothioate (Petersen et al. 1991 ). Our findings also suggest a possible mechanism for the regulation of the frequency of [Ca2+]i oscillations in cells containing IP3-sensitive Ca2+ stores. One test of the value of our findings will come from future studies that extend these observations to other cell types and incorporate these mechanisms into mathematical models of [Ca2+]i signaling.

Since platelets express primarily the type 1 isoform of the IP3-R (O'Rourke et al. 1995 ; Quinton and Dean 1996 ) and megakaryocytes are the precursors of platelets, our findings may directly reflect properties of the type 1 IP3-R. Remember that, as mentioned in the INTRODUCTION, cerebellar microsomes (Joseph et al. 1989 ; Combettes et al. 1994 ; Hannaert-Merah et al. 1995 ) and permeabilized A7r5 smooth muscle cells (Bootman et al. 1995 ), which contain primarily the type 1 isoform of the IP3-R, exhibit decreased Ca2+-dependent inhibition at elevated IP3 concentrations. Moreover, single channel recordings from the cerebellar type 1 IP3-R (Kaftan et al. 1997 ) and a similar receptor found in Xenopus oocytes (Mak et al. 1998 ) indicate that the open probability remains high in the presence of a saturating level of IP3, even if [Ca2+]i is raised to high concentrations. It should be kept in mind that IP3 binding to the purified cerebellar type 1 IP3-R is not inhibited by Ca2+ and it was proposed that inhibition by Ca2+ required an accessory protein (Supattapone et al. 1988 ; Benevolensky et al. 1994 ), which was recently shown to be calmodulin (Michikawa et al. 1999 ) (Fig 9).

The observation that Ca2+-dependent inhibition of the type 1 IP3-R is mediated by calmodulin implies that inhibition of calmodulin should disinhibit Ca2+-dependent inhibition of IP3-mediated Ca2+ release (Michikawa et al. 1999 ). Based on our findings, we would predict that such a disinhibition would transform a [Ca2+]i oscillation into a more sustained [Ca2+]i elevation (for example, see Fig 6). This experiment has in fact already been done in rat megakaryocytes, where it was found that the calmodulin inhibitors W-7 and trifluoperazine caused the agonist-induced [Ca2+]i oscillation to become a more sustained [Ca2+]i elevation (Uneyama et al. 1993 ). Note that W-7 is the same calmodulin inhibitor used in the study of Michikawa et al. 1999 . One test of the worthiness of our interpretation of these findings will come from the extension of these observations to other cell types.

Whether or not these properties of the type I receptor also belong to the type II and III IP3-Rs is problematic. Recent single-channel bilayer recordings from the type II and III receptors indicate that they do not exhibit Ca2+-dependent inhibition (Hagar et al. 1998 ; Ramos-Franco et al. 1998 ); however, in bilayer recordings, essential accessory proteins may have been lost. On the other hand, Ca2+-dependent inhibition has been observed, using other techniques, in some cell types that contain primarily the type II and III receptors (Taylor 1998 ); however, these studies are complicated by the presence of other receptor subtypes. Further experimental work will be needed to determine the extent to which the findings presented here are exemplary of cells that contain primarily the type II and III IP3-Rs. It may be that cells contain mixtures of the different isoforms of the IP3-R to combine properties specific to each type of receptor.

One of the striking features of IP3-mediated Ca2+ release in megakaryocytes is the highly nonlinear dependence between IP3 and peak Ca2+ (Fig 1 and Fig 4). In other cell types, the dependence is not as steep (Khodakhah and Ogden 1995 ; Oancea and Meyer 1996 ; Carter and Ogden 1997 ; Ogden and Capiod 1997 ); for example, in rat basophilic leukemia cells, the Hill coefficient is 3.2, as compared with 7 for megakaryocytes. There are two factors that would be expected to contribute to the nonlinear dependence between IP3 and peak Ca2+. First is a requirement for the binding of several IP3 molecules to the IP3-receptor before the channel can open, and second is an amplification of Ca2+ release by positive feedback mediated by Ca2+ (for example, see Iino 1990 ; Bezprozvanny et al. 1991 ). It may be that there are additional unknown factors at work in megakaryocytes, which are responsible for the exceptionally steep dependence found in these cells.

It might be argued that as the result of inhibition of the 5-phosphatase by 2,3-DPG, more IP3 is converted by the IP3-3-kinase to inositol 1,3,4,5-tetrakisphosphate (IP4). IP4 has been shown to enhance the amount of Ca2+ mobilized by submaximal concentrations of IP3 in the L1210 cell line (Loomis-Husselbee et al. 1996 , Loomis-Husselbee et al. 1998 ). If such a phenomenon were to occur in megakaryocytes, it could possibly explain our findings with 2,3-DPG; however, to our knowledge, it is not known whether such a phenomenon occurs in megakaryocytes or for that matter in cells other than L1210 cells. Furthermore, such a mechanism would not be able to explain our findings with GPIP2. Moreover, it should be kept in mind that it is still controversial whether or not IP4 plays any role in Ca2+ signaling (Irvine 1992 ; Putney and Bird 1993 ).

Although the findings reported here were obtained in megakaryocytes, they should be relevant to calcium mobilization in platelets also; in as much as megakaryocytes are the precursors of platelets. Specifically, we speculate that our findings suggest a role for pleckstrin, which is a major substrate for PKC in platelets, in regulating [Ca2+]i oscillations by regulating the lifetime of IP3.


  Footnotes

Dr. Tertyshnikova's present address is Bristol-Myers Squibb Co., Pharmaceutical Research Institute, Wallingford, CT 06492-7660.
1 Abbreviations used in this paper: IP3, inositol 1,4,5-trisphosphate; IP3-R, IP3 receptor.


  Acknowledgements
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

We thank Drs. L. Jaffe, R. Shaafi, M. Terasaki, and J. Watras for their constructive criticisms of an earlier version of this manuscript.

Submitted: 29 September 1999
Revised: 22 February 2000
Accepted: 28 February 2000


  References
Top
Abstract
INTRODUCTION
METHODS
RESULTS
DISCUSSION
Acknowledgements
References

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