Voltage-dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors

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Voltage-dependent Ca²⁺ release in rat megakaryocytes requires functional IP₃ receptors

Michael J. Mason and Martyn P. Mahaut-Smith

Department of Physiology, University of Cambridge, Cambridge CB2 3EG, UK

MS 11978 Received 24 November 2000; accepted after revision 16 January 2001

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Using simultaneous whole-cell patch-clamp and fluorescence measurements of [Ca²⁺]_i in rat megakaryocytes we have investigated the requirement for functional inositol 1,4,5-trisphosphate (IP₃) receptors in Ca²⁺ release induced by membrane depolarization during agonist stimulation.
Voltage-dependent Ca²⁺ release was observed during application of the IP₃-generating agonists U46619 (a thromboxane A₂ analogue) and ADP. Furthermore, voltage-dependent Ca²⁺ release was observed in the absence of exogenous agonist following sensitization of IP₃ receptors with thimerosal.
Depolarization-induced Ca²⁺ release was not detected during depletion of intracellular Ca²⁺ stores by thapsigargin. Thus, depletion of stores alone is not sufficient to confer voltage dependence upon the Ca²⁺ release mechanism.
Block of IP₃ receptors by carbacyclin-stimulated elevations in cAMP, uncaging of cAMP or exposure to a high concentration of caffeine reversibly abolished Ca²⁺ increases stimulated by both ADP and depolarization.
The cAMP-dependent block was prevented by a peptide inhibitor of protein kinase A, indicating that an alteration of adenylate cyclase activity leading to modulation of protein kinase A activity does not underlie the control of Ca²⁺ release by voltage.
These results are consistent with the requirement for functional IP₃ receptors for voltage control of Ca²⁺ release from intracellular stores during inositol lipid signalling. The data also indicate the involvement of a voltage sensor downstream of surface membrane receptors in the depolarization-evoked Ca²⁺ response.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Depolarization of the plasma membrane is an important means by which some cells modulate Ca²⁺ release from intracellular stores. This occurs directly in skeletal muscle by a configurational coupling mechanism between the T-tubular voltage-dependent Ca²⁺ channel and the ryanodine receptor. In other cells, for example cardiac myocytes, the Ca²⁺ release induced by depolarization requires Ca²⁺ influx and subsequent Ca²⁺-induced Ca²⁺ release (CICR) (Schneider & Chandler, 1973; Fabiato, 1983; Rios & Brum, 1987). Inositol 1,4,5-trisphosphate (IP₃)-dependent Ca²⁺ release is also subject to membrane potential-dependent modulation as a consequence of altered Ca²⁺ influx and the modulation of IP₃ receptor function by Ca²⁺ (Iino, 1990; Bezprozvanny et al. 1991; Finch et al. 1991). An increase in [Ca²⁺]_i can activate and inactivate IP₃ receptors, depending upon the concentration and IP₃ receptor isoform (Bezprozvanny et al. 1991; Taylor & Traynor, 1995; Hagar et al. 1998; Moraru et al. 1999). Additionally, phospholipase C is known to be activated by an increase in [Ca²⁺]_i (Eberhard & Holz, 1988).

The control of IP₃-dependent Ca²⁺ release by cell membrane potential may not be restricted to an effect via modulation of cytosolic [Ca²⁺]. We have recently reported in the rat megakaryocyte that activation of purinoceptors linked to inositol lipid signalling induces a marked sensitivity of the Ca²⁺ release process to changes in membrane potential (Mahaut-Smith et al. 1999; Mason et al. 2000). Depolarization stimulates Ca²⁺ release from intracellular stores whilst hyperpolarization results in a net Ca²⁺ reuptake. The megakaryocyte is a non-excitable cell type in which there is no evidence for ryanodine receptors or CICR (Uneyama et al. 1993). The conclusion that the effect of changes in membrane potential on [Ca²⁺]_i is a result of modulation of Ca²⁺ release from intracellular stores arises from the fact that (1) the changes in [Ca²⁺]_i are observed in the absence of extracellular Ca²⁺, thus ruling out modulation of a Ca²⁺ influx pathway (Mahaut-Smith et al. 1999; Mason et al. 2000), and (2) similar effects of changes in membrane potential are observed under conditions in which Na⁺-Ca²⁺ and Na⁺-Ca²⁺, K⁺ exchange activity is abolished, thus ruling out the possibility of membrane potential modulation of the activity of these exchangers in the observed response (Mahaut-Smith et al. 1999; Mason et al. 2000).

A similar bipolar dependence of the cellular Ca²⁺ stores on membrane voltage has also been detected in coronary artery smooth muscle cells during activation of muscarinic ACh receptors (Ganitkevich & Isenberg, 1993). The underlying mechanism may therefore represent a general means of controlling receptor-dependent Ca²⁺ release via the membrane potential. In both megakaryocytes and smooth muscle, intracellular dialysis with heparin, an IP₃ receptor antagonist (Brezprozvanny & Ehrlich, 1994), blocked the voltage-dependent response, providing preliminary evidence for an involvement of functional IP₃ receptors. The caveats to this use of heparin are the inability to reverse its application and the recognized effects of heparin on additional cellular processes (Ito et al. 1990), for example, on impairment of adenylate cyclase activation (Reches et al. 1979; Cutler & Christian, 1984). The present experiments were undertaken to define directly the requirement for functional IP₃ receptors in the voltage control of Ca²⁺ release from intracellular stores in the rat megakaryocyte. We provide evidence that functional IP₃ receptors are required for membrane potential modulation of Ca²⁺ release and that voltage control of Ca²⁺ release can occur independently from plasma membrane receptor stimulation, thus indicating the existence of a voltage sensor downstream of the surface receptor. This may represent a more general mechanism by which electrogenic and inositol lipid signalling pathways can interact.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Cell isolation

Male Wistar rats (150-300 g) were killed by exposure to a rising concentration of CO₂ followed by cervical dislocation, and megakaryocytes were isolated from femoral and tibial marrow as described previously (Mahaut-Smith et al. 1999). Apyrase Type V or VII (0.16-0.32 U ml^-1) (Sigma-Aldrich, Poole, UK) was present during cell preparation but omitted during experiments.

Solutions

The standard external saline contained (mM): 145 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 10 Hepes, 10 D-glucose; titrated to pH 7.35 with NaOH. The pipette saline contained (mM): 150 KCl, 2 MgCl₂, 0.1 EGTA, 0.05 Na₂GTP, 10 Hepes, adjusted to pH 7.2 with KOH, and either 0.05 K₅fura-2 or 0.05 (NH₄)₅fluo-3. ADP, the thromboxane A₂ analogue U46619, thimerosal and dithiothreitol were obtained from Sigma-Aldrich. 4,5-Dimethoxy-2-nitrobenzyl-caged cAMP (DMNB-caged cAMP) and K₅fura-2 were purchased from Molecular Probes (Eugene, OR, USA) while the 2-nitrophenyl ethyl ester of D-myo-inositol 1,4,5-trisphosphate (NPE-caged IP₃) and PKI 6-22 (an inhibitory peptide of the catalytic subunit of protein kinase A) were obtained from Calbiochem-Novabiochem (Nottingham, UK). (NH₄)₅fluo-3 was purchased from Calbiochem-Novabiochem or Molecular Probes.

Electrical recording

Conventional whole-cell patch-clamp recordings were carried out using an Axopatch 200A amplifier with CV202 headstage (Axon Instruments, USA) in voltage-clamp mode. pCLAMP 6 software and a Digidata 1200 interface (Axon Instruments) were used to generate voltage steps; 70-75 % series resistance compensation was achieved. Membrane potentials have not been corrected for the small offset (approximately -3 mV) that results from the liquid-liquid junction potential between internal and external saline solutions.

Fluorescence measurements

A Cairn Spectrophotometer System (Cairn Research Ltd, Kent, UK) coupled to a Nikon Diaphot TMD inverted microscope (Nikon, Japan) was used to measure fura-2 or fluo-3 fluorescence during simultaneous whole-cell patch clamp (see Mahaut-Smith, 1998, for further details). Excitation wavelengths were 340 and 380 nm for fura-2 and 490 nm for fluo-3. A 400 nm (fura-2) or 503 nm (fluo-3) dichroic mirror was used to separate the excitation and emission light. The emission bandwidth was 480-600 nm for fura-2 and 528-600 nm for fluo-3. Fluorescence emission was detected by a photomultiplier tube and collected together with the electrophysiological signals, using Cairn fluorescence software. Data were sampled at 60 Hz, averaged to give a final acquisition rate of 15 Hz and exported for analysis within IGOR (Wavemetrics, Lake Oswego, OR, USA) or Microcal Origin (Microcal Software Inc., Northampton, MA, USA). For presentation, a Gaussian filter within IGOR was applied to the data from certain experiments. For calibration, fura-2 constants, R_min (fluorescence ratio in the absence of Ca²⁺) and R_max (fluorescence ratio in the presence of saturating Ca²⁺), were obtained extracellularly since it was difficult to clamp [Ca²⁺]_i at high levels in the megakaryocyte. A calibration kit (Molecular Probes) was used to derive a K_d for fura-2 (258 nM). After application of a viscosity correction factor (0.85) to R_min and R_max (Poenie, 1990), background-corrected 340 nm/380 nm values were converted to [Ca²⁺]_i as described by Grynkiewicz et al. (1985). Single wavelength fluo-3 data are presented as raw, background-corrected fluorescence.

Flash photolysis of caged compounds

Cells were loaded with caged cAMP by incubation for a minimum of 2 h with 200 µM of the membrane-permeant DMNB derivative of caged cAMP. Cells were loaded with the NPE derivative of caged IP₃ by dialysis from the patch pipette containing 100 µM caged IP₃. Uncaging was achieved using a Cairn Flash Photolysis unit. The flash illumination from a high-intensity xenon arc lamp (Advance Radiation, Inc., Santa Clara, CA, USA) was filtered by a broad bandpass UV filter (UG5, Comar Instruments, Cambridge, UK). A 503 nm dichroic mirror with extended UV reflectance (Chroma Technology Corp., Brattleboro, VT, USA), mounted in the epifluorescence port, allowed simultaneous delivery of uncaging and 490 nm light for excitation of fluo-3. The flash duration was approximately 1 ms and the flash intensity was adjusted by controlling the level of capacitance charged at 400 V on the flash photolysis unit. No attempts were made to estimate the quantity of cAMP or IP₃ released during each flash.

Solution application

Application of extracellular agonists and antagonists was by gravity-driven bath superfusion. Solution changes, as noted in the figures, have been corrected for delays resulting from dead space within the superfusion system. All experiments were performed at room temperature (20-25 °C).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

We have previously reported that release of Ca²⁺ from intracellular stores within the rat megakaryocyte becomes highly sensitive to changes in membrane potential during exposure to ADP (Mahaut-Smith et al. 1999; Mason et al. 2000). To date, depolarization-induced Ca²⁺ release in the megakaryocyte has only been demonstrated in the presence of the IP₃-generating agonist ADP. Experiments were undertaken to determine whether voltage-dependent Ca²⁺ release was a more general phenomenon associated with inositol lipid signalling. To address this hypothesis directly we have investigated the effect of membrane depolarization during exposure to U46619, a stable thromboxane A₂ analogue. In megakaryocytic cell lines and platelets, U46619 has been shown to stimulate membrane phospholipid turnover and an increase in [Ca²⁺]_i (Dorn & Davis, 1992). In the rat megakaryocyte, application of 300 nM U46619 resulted in elevations in [Ca²⁺]_i that took the form of either repetitive Ca²⁺ oscillations or a Ca²⁺ transient followed by a sustained plateau phase of elevated [Ca²⁺]_i (Fig. 1A). Depolarization to 0 mV during application of U46619 resulted in elevations in [Ca²⁺]_i and frequently stimulated Ca²⁺ oscillations in non-oscillating cells (Fig. 1A). Transient elevations in [Ca²⁺]_i in response to depolarization to 0 mV were observed in 12 of 12 cells. Repolarization to -75 mV was accompanied by a marked decline in [Ca²⁺]_i. Following removal of U46619, depolarization to 0 mV had no effect upon [Ca²⁺]_i while similar effects of depolarization and repolarization on [Ca²⁺]_i were observed during subsequent exposure to ADP, as previously reported (Mahaut-Smith et al. 1999; Mason et al. 2000). A similar depolarization-evoked [Ca²⁺]_i increase in the presence of U46619 was observed in Ca²⁺-free saline in 9 of 9 cells (Fig. 1B), indicating that this response is predominantly due to release of Ca²⁺ from intracellular stores, as previously reported for ADP (Mahaut-Smith et al. 1999; Mason et al. 2000). Furthermore, elevations in [Ca²⁺]_i induced by depolarization did not arise from alterations in Na⁺-Ca²⁺ or Na⁺-Ca²⁺, K⁺ exchange activity as depolarization-induced Ca²⁺ increases were detected in the presence of 5 mM Ni²⁺, an inhibitor of both exchangers (Fig. 1C) (Hinde et al. 1999; Kimura et al. 1999). In these experiments, inhibition by Ni²⁺ of the voltage-dependent K⁺ channel was monitored to confirm adequate superfusion of the cell with extracellular Ni²⁺ (Fig. 1C, middle panel).

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Figure 1. Depolarization-dependent Ca²⁺ release during application of U46619 and ADP
Single megakaryocytes were whole-cell patch clamped and loaded with fura-2 via dialysis from the patch pipette. In A, 300 nM U46619 or 1 µM ADP was applied extracellularly where indicated. In B, the experiment was performed in nominally Ca²⁺-free solution. The cell was superfused with nominally Ca²⁺-free solution for 90 s prior to addition of U46619. In C, the cell was superfused with 300 nM U46619 in the presence of 5 mM Ni²⁺. The whole-cell membrane current is shown (middle panel) and documents Ni²⁺ block of the voltage-dependent K⁺ current. In A-C, voltage steps between -75 and 0 mV were applied where indicated. V_m, membrane potential.

These data support the hypothesis that the ability of membrane potential to modulate release of Ca²⁺ from intracellular stores is a more general phenomenon associated with inositol lipid signalling rather than being solely associated with ADP stimulation. To further investigate this hypothesis, experiments were undertaken to determine whether sensitization of the IP₃ receptor with the thiol reagent thimerosal (Bootman et al. 1992; Hilly et al. 1993; Sayers et al. 1993) could confer membrane potential sensitivity upon Ca²⁺ release in the absence of exogenously applied agonist. Exposure of an unstimulated rat megakaryocyte to 100 µM thimerosal induced non-periodic Ca²⁺ transients after a delay of approximately 60-70 s (Fig. 2A). This effect of thimerosal has been ascribed to an increased sensitivity of the IP₃ receptor such that basal levels of production of IP₃ can stimulate Ca²⁺ release (Bootman et al. 1992; Hilly et al. 1993; Sayers et al. 1993). In the presence of thimerosal, depolarizations to 0 mV stimulated a transient increase in [Ca²⁺]_i, similar to the depolarization-evoked response observed during agonist application (e.g. Fig. 1). The changes in [Ca²⁺]_i stimulated by thimerosal could be fully reversed by combined removal of thimerosal and application of 1 mM of the reducing agent dithiothreitol (DTT), as previously reported in HeLa cells (Bootman et al. 1992). DTT also reversed the effect of depolarization on [Ca²⁺]_i. A second application of 100 µM thimerosal tended to induce a more sustained increase in [Ca²⁺]_i, and depolarization also induced prolonged increases in [Ca²⁺]_i in contrast to the transient responses earlier in the experiment. Depolarization induced a [Ca²⁺]_i increase in 12 of 13 cells following sensitization of the IP₃ receptor by thimerosal, and in each case, the response was reversed by DTT. The one cell in which depolarization failed to influence [Ca²⁺]_i also showed no response to the thiol reagent alone. The depolarization-evoked [Ca²⁺]_i increase in the presence of thimerosal also involved release of Ca²⁺ from intracellular stores, since similar responses were observed in 8 of 8 cells in nominally Ca²⁺-free saline (Fig. 2B).

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Figure 2. Effect of depolarization on [Ca²⁺]_i during thimerosal and thapsigargin treatment
A-C, single megakaryocytes were whole-cell patch clamped and loaded with fura-2 via dialysis from the patch pipette. Where indicated, 100 µM thimerosal, 1 mM DTT or 1 µM thapsigargin was added. The experiment presented in B was performed in nominally Ca²⁺-free solution. The cell was superfused with nominally Ca²⁺-free solution for 200 s prior to addition of thimerosal. In A-C, voltage steps between -75 and 0 mV were applied where indicated.

While the increase in [Ca²⁺]_i induced by depolarization in the presence of thimerosal most probably reflects the increased IP₃ sensitivity of the endosomal IP₃ receptor (Bootman et al. 1992; Hilly et al. 1993; Sayers et al. 1993), it has also been demonstrated that this thiol alkylating agent inhibits endosomal Ca²⁺-ATPase activity (Bootman et al. 1992). This secondary effect may underlie the more sustained increases in [Ca²⁺]_i observed during the second application of thimerosal in Fig. 2A. In the experiment shown in Fig. 2C, we investigated whether store depletion alone could induce voltage dependence using the endosomal Ca²⁺-ATPase inhibitor thapsigargin. Application of thapsigargin to a megakaryocyte held at -75 mV resulted in a slow increase in [Ca²⁺]_i as judged by an increase in the 340 nm/380 nm fura-2 fluorescence ratio. The cell was bathed in Ca²⁺-containing saline, therefore the elevation in [Ca²⁺]_i will be derived from both Ca²⁺ release from stores and the activation of store-dependent Ca²⁺ influx (Somasundaram & Mahaut-Smith, 1994). Depolarization to 0 mV at several time points throughout the initial stages of store depletion failed to accelerate the [Ca²⁺]_i increase. In fact, as the stores became progressively more depleted, depolarization resulted in a decrease in [Ca²⁺]_i and repolarization produced an [Ca²⁺]_i increase. This can be explained by a progressive increase in the contribution of store-dependent Ca²⁺ influx to the thapsigargin-evoked Ca²⁺ signal. Depolarization to 0 mV greatly reduces the Ca²⁺ influx as a consequence of reduced driving force and the underlying inward rectification of the membrane conductance (I_crac; Ca²⁺ release-activated Ca²⁺ current) responsible for store-dependent Ca²⁺ influx in the megakaryocyte (Somasundaram & Mahaut-Smith, 1994). Therefore, depolarization during depletion of Ca²⁺ stores is not sufficient by itself to confer voltage sensitivity on Ca²⁺ release. Thus, sensitization of IP₃ receptors most probably accounts for the induction of depolarization-evoked Ca²⁺ release during thimerosal treatment.

In order to more directly investigate the involvement of IP₃ receptors in this novel voltage-dependent Ca²⁺ release mechanism, we have established conditions in our whole-cell patch-clamp recordings that reversibly block IP₃-dependent Ca²⁺ release. Superfusion with 100 nM carbacyclin (n = 4; Fig. 3A) or 10 mM caffeine (n = 5; Fig. 3C) was found to reversibly abolish the Ca²⁺ release induced by repetitive uncaging of IP₃. Carbacyclin, a stable prostacyclin analogue, has been shown to inhibit IP₃ receptors via an elevation of cAMP (Tertyshnikova & Fein, 1998, and references therein), whereas IP₃ receptors are inhibited directly by high concentrations of caffeine (Wakui et al. 1990; Parker & Ivorra, 1991; Brown et al. 1992; Bezprozvanny et al. 1994). In separate experiments, the effect of inhibiting IP₃ receptors was tested on the depolarization-induced Ca²⁺ release observed during ADP application. ADP (1 µM) evoked multiple oscillations of [Ca²⁺]_i, which were arrested by either carbacyclin (Fig. 3B) or caffeine (Fig. 3D). Importantly, depolarization from -75 to 0 mV had no effect on [Ca²⁺]_i in 9 of 9 cells in the presence of carbacyclin (Fig. 3B) and depolarization from -75 to 0 mV had no effect on [Ca²⁺]_i in 6 of 7 cells in the presence of caffeine (Fig. 3D). In the one cell that showed a voltage-dependent increase in [Ca²⁺]_i, caffeine failed to completely abolish ADP-induced [Ca²⁺]_i oscillations. The parallel inhibition of outward Ca²⁺-dependent K⁺ current (Fig. 3C) confirmed that the effect of caffeine was not via a direct alteration of indicator fluorescence (Muschol et al. 1999). Following removal of either carbacyclin or caffeine, depolarization evoked a clear [Ca²⁺]_i increase (Fig. 3B and D), which we have previously shown to result primarily from Ca²⁺ release from intracellular stores (Mahaut-Smith et al. 1999; Mason et al. 2000). Taken in concert, the inhibitory actions of carbacyclin and caffeine are consistent with a requirement for functional IP₃ receptors for depolarization to evoke Ca²⁺ release from intracellular stores in the megakaryocyte.

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Figure 3. Carbacyclin and caffeine inhibit IP₃-, ADP- and depolarization-dependent Ca²⁺ release from intracellular stores
Single rat megakaryocytes were loaded with fluo-3 (A-C) or fura-2 (D) and 100 µM caged IP₃ (A and C) via dialysis from the patch pipette during whole-cell recording. At each arrow in A and C, IP₃ was elevated by an identical flash of UV light. ADP, carbacyclin and caffeine were added where indicated. The cells were held at a constant holding potential (HP) of -75 (A) or -60 mV (C). The bottom panel in C shows the whole-cell current. In B and D, voltage steps between -75 and 0 mV were applied as indicated.

Inhibition by carbacyclin of IP₃-stimulated Ca²⁺ release has been shown to result from an elevation of cAMP and the inhibition of IP₃ receptors by cAMP-dependent protein kinase A (PKA) (Tertyshnikova & Fein, 1998). In the platelet, ADP is known to activate receptors coupled to adenylate cyclase (Kunapuli & Daniel, 1998), therefore we considered the possibility that voltage-dependent modulation of cAMP levels, leading to modulation of PKA activity, was responsible for the voltage-dependent Ca²⁺ release. Initially, conditions were established whereby flash photolysis of cytosolic caged cAMP could effectively abolish IP₃-mediated Ca²⁺ release, as judged by the complete block of ADP-dependent Ca²⁺ signalling (see Fig. 4A and Methods for full descriptions of caged cAMP loading). Release of caged cAMP inhibited the [Ca²⁺]_i oscillations in response to 1 µM ADP in 43 of 47 cells. This inhibition reversed after 10-15 s, which most probably reflects the degradation of cAMP by endogenous phosphodiesterase activity. Importantly, in the cell shown in Fig. 4A, depolarization to 0 mV immediately after flash release of cAMP had no effect upon [Ca²⁺]_i, whereas depolarizations evoked marked transient increases in [Ca²⁺]_i in the same cell during active ADP-dependent Ca²⁺ signals (Fig. 4A). Complete inhibition of depolarization-evoked [Ca²⁺]_i increases immediately after flash release of cAMP were observed in 29 of 34 cells. In the five cells in which uncaging light was not completely effective, ADP-evoked Ca²⁺ signals were also not completely abolished. Therefore, reduced loading during the preincubation period or dialysis of the caged compound during whole-cell recordings is the most likely explanation for the lack of complete inhibition in a small proportion of cells. As shown previously (Tertyshnikova & Fein, 1998), an inhibitory peptide of the protein kinase A catalytic subunit (PKI 6-22; Glass et al. 1989) reversed the inhibitory effects of cAMP on ADP-induced [Ca²⁺]_i oscillations (Fig. 4B) while having no observable effect upon the ADP-induced Ca²⁺ oscillations. Interestingly, in the presence of PKI 6-22, depolarization was also able to evoke a Ca²⁺ increase immediately after uncaging of cAMP and at other times during the whole-cell recording (n = 10; Fig. 4B). These data are consistent with the hypothesis that the inhibitory effect of cAMP on depolarization-mediated Ca²⁺ release results from activation of PKA and subsequent inhibition of IP₃ receptors. These data also indicate that modulation of adenylate cyclase leading to control of PKA activity is not responsible for the effect of voltage on IP₃ receptor-dependent Ca²⁺ release in the megakaryocyte.

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Figure 4. PKA is required for the inhibitory action of cAMP on ADP- and depolarization-induced release of Ca²⁺ from intracellular stores
A and B, single megakaryocytes were loaded with caged cAMP by incubation for 2 h with the membrane-permeant DMNB derivative, whole-cell patch clamped and loaded with fluo-3 via dialysis from the patch pipette. Where indicated, cAMP was elevated by a flash of UV light. In B, 100 µM PKI 6-22, an inhibitory peptide of the catalytic subunit of PKA, was included in the patch pipette. In A and B, voltage steps between -75 and 0 mV were applied where indicated.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

During purinoceptor stimulation of rat megakaryocytes with ADP, membrane potential changes over the physiological range markedly alter Ca²⁺ release from intracellular stores (Mahaut-Smith et al. 1999; Mason et al. 2000). We have previously reported that this effect of depolarization is inhibited by heparin, consistent with a requirement for functional IP₃ receptors (Mahaut-Smith et al. 1999). However, heparin has been shown to target other signalling molecules and cell processes (Ito et al. 1990), raising questions as to its site of action. The ability of carbacyclin and caffeine to reversibly inhibit both IP₃-dependent Ca²⁺ release and depolarization-evoked Ca²⁺ release (Fig. 3) is consistent with the requirement for functional IP₃ receptors in the control of Ca²⁺ release by membrane potential. Carbacyclin has been shown to inhibit IP₃ receptors via activation of receptors coupled to adenylate cyclase, elevation of cAMP and activation of PKA (Tertyshnikova & Fein, 1998). Inhibition of depolarization-evoked Ca²⁺ increases by carbacyclin also occurred via this pathway since voltage-dependent Ca²⁺ increases were reversibly blocked following direct elevation of cAMP by flash photolysis (Fig. 4A) and this block was prevented by inclusion in the patch pipette of PKI 6-22, an inhibitory peptide of the PKA catalytic subunit (Fig. 4B). The ability to observe the ADP-induced depolarization-dependent Ca²⁺ increase in the presence of a blocker of PKA activity suggests that modulation by the transmembrane potential of adenylate cyclase activity leading to cAMP-dependent regulation of PKA activity does not underlie the effect of voltage on intracellular Ca²⁺ stores. These results, in concert with the previously reported block by heparin, are consistent with the hypothesis that the depolarization-evoked Ca²⁺ increase observed during receptor-mediated inositol lipid signalling requires functional IP₃ receptors.

The existence of a mechanism whereby the cell membrane potential can control IP₃ receptor-dependent Ca²⁺ release has important implications for the control of Ca²⁺ signalling in excitable and non-excitable tissues. To date, direct evidence for voltage regulation of IP₃ receptor-dependent Ca²⁺ release, without an involvement of Ca²⁺ influx, has been provided in the rat megakaryocyte (Mahaut-Smith et al. 1999; Mason et al. 2000), and in smooth muscle from coronary artery (Ganitkevich & Isenberg, 1993) or gastric pylorus (Van Helden et al. 2000). Activation of purinoceptors and thromboxane A₂ receptors in the megakaryocyte, and muscarinic cholinergic receptors in coronary artery myocytes, induced the voltage-dependent Ca²⁺ release mechanism, whereas in the gastric smooth muscle, depolarization was able to release Ca²⁺ during spontaneous/pacemaker activity. Despite these wide-ranging conditions, the presence of the response in other tissues remains to be established.

At present, the nature of the voltage sensor responsible for coupling changes in plasma membrane potential to the status of the intracellular Ca²⁺ stores is unclear. One hypothesis, as proposed by Ganitkevich & Isenberg (1993) in smooth muscle, is that membrane potential influences one or more stages in the inositol lipid signalling cascade from receptor activation to production of IP₃. Importantly, in the rat megakaryocyte and the coronary smooth muscle cell, voltage dependence of Ca²⁺ release could be observed independent of receptor activation, consistent with a requirement for a voltage sensor downstream of the plasma membrane receptor.

A second hypothesis that can potentially account for the voltage dependence is that substrate availability at receptors and/or enzymes is altered by the voltage field. Several molecules required for, or consumed within, the signalling cascade leading to IP₃ production are highly polar, including extracellular ADP, intracellular GTP and phosphatidylinositol 4,5-bisphosphate. Therefore, the membrane voltage may affect their binding and consequently the efficacy of IP₃ generation. The effect of thimerosal indicates that activation of surface receptors, and therefore voltage-dependent movements of extracellular agonist, are not essential to the mechanism in the megakaryocyte.

The two theories proposed above suggest that membrane potential can alter the level of IP₃ generation by phospholipase C and thus explain the absolute requirement for IP₃ receptors demonstrated in the present study. Direct measurements of IP₃ production under voltage-clamp conditions have not been reported at the single cell level. However, in multi-cell preparations, hyperpolarization caused a reduction in agonist-evoked IP₃ production in smooth muscle cells from rabbit mesenteric artery (Itoh et al. 1992), while depolarization stimulated IP₃ generation in cultured skeletal muscle (Jaimovich et al. 2000).

A third hypothesis that could account for the voltage dependence is a conformational coupling model, in which protein-protein interactions between plasma and intracellular Ca²⁺ storage membranes control Ca²⁺ release. A situation where configurational coupling is well established to occur is the interaction between T-tubule dihydropyridine receptors and ryanodine receptors on the Ca²⁺ store, which controls the release of Ca²⁺ during excitation-contraction coupling in skeletal muscle (Schneider & Chandler, 1973). A conformational coupling hypothesis has also been proposed to account for the gating of a Ca²⁺ influx pathway by depletion of intracellular Ca²⁺ stores in many cells (Irvine, 1990). Experimental evidence has recently been provided for this type of coupling in which store-dependent Ca²⁺ channels are controlled by IP₃ receptors (Kiselyov et al. 1998; Boulay et al. 1999; Ma et al. 2000). Thus, IP₃ receptors may have multiple roles and we cannot rule out the possibility that the requirement of the receptor in our experiments results from an action within a configurational coupling site.

The present experiments have provided evidence consistent with the conclusion that IP₃ receptors play a fundamental role in the ability of membrane depolarization to stimulate release of Ca²⁺ from intracellular stores in the rat megakaryocyte during inositol lipid signalling. Modulation of IP₃-dependent Ca²⁺ release by membrane voltage has important implications for the control of inositol lipid signalling by ionotropic events and may have far reaching implications for the control of Ca²⁺ signalling in other cell types, particularly excitable cells that experience wide fluctuations in membrane potential.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

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BEZPROZVANNY I., WATRAS, J. & EHRLICH, B. E. (1991). Bell-shaped calcium-response curves of Ins(1,4,5)P₃ and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751-754 [Medline]

BOOTMAN M. D., TAYLOR, C. W. & BERRIDGE, M. J. (1992). The thiol reagent, thimerosal, evokes Ca²⁺ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor. Journal of Biological Chemistry 267, 25113-25119 [Abstract]

BOULAY G., BROWN, D. M., QIN, N., JIANG, M., DIETRICH, A., ZHU, M. X., CHEN, Z., BIRNBAUMER, M., MIKOSHIBA, K. & BIRNBAUMER, L. (1999). Modulation of Ca²⁺ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca²⁺ entry. Proceedings of the National Academy of Sciences of the USA 96, 14955-14960 [Abstract/Full Text]

BROWN G. R., SAYERS, L. G., KIRK, C. J., MICHELL, R. H. & MICHELANGELI, F. (1992). The opening of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ channel in rat cerebellum is inhibited by caffeine. Biochemical Journal 282, 309-312 [Medline]

CUTLER L. S. & CHRISTIAN, C. P. (1984). Inhibition of rat salivary gland adenylate cyclase by glycosaminoglycans and high molecular weight polyanions. Archives of Oral Biology 29, 629-633 [Medline]

DORN W. G. II_. & DAVIS, M. G. (1992). Differential megakaryocytic desensitization to platelet agonists. American Journal of Physiology 263, C864-872 [Medline]

EBERHARD D. A. & HOLZ, R. W. (1988). Intracellular Ca²⁺ activates phospholipase C. Trends in Neurosciences 12, 517-520

FABIATO A. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. American Journal of Physiology 245, C1-14 [Medline]

FINCH E. A., TURNER, T. J. & GOLDIN, S. M. (1991). Calcium as a co-agonist of inositol 1,4,5-trisphosphate-induced calcium release. Science 252, 443-446 [Medline]

GANITKEVICH V. YA. & ISENBERG, G. (1993). Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca²⁺ transients in guinea-pig coronary myocytes. Journal of Physiology 470, 35-44 [Abstract]

GLASS D. B., CHENG, H. C., MENDE-MEULLER, L., REED, J. & WALSH, D. A. (1989). Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein. Journal of Biological Chemistry 264, 8802-8810 [Abstract]

GRYNKIEWICZ G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450 [Abstract]

HAGAR R. E., BURGSTAHLER, A. D., NATHANSON, M. H. & EHRLICH, B. E. (1998). Type III InsP₃ receptor channel stays open in the presence of increased calcium. Nature 396, 81-84 [Medline]

HILLY M., PIÉTRI-ROUXEL, F., COQUIL, J. F., GUY, M. & MAUGER, J. P. (1993). Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor. Journal of Biological Chemistry 268, 16488-16494 [Abstract]

HINDE A. K., PERCHENET, L., HOBAI, I. A., LEVI, A. J. & HANCOX, J. C. (1999). Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 °C, dialysed internally with cAMP-free and cAMP-containing solutions. Cell Calcium 25, 321-331 [Medline]

IINO M. (1990). Biphasic Ca²⁺ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. Journal of General Physiology 95, 1103-1122 [Medline]

IRVINE R. F. (1990). 'Quantal' Ca²⁺ release and the control of Ca²⁺ entry by inositol phosphates - a possible mechanism. FEBS Letters 263, 5-9 [Medline]

ITO H., TAKIKAWA, R., IGICHI, M., HAMADA, E., SUGIMOTO, T. & KURACHI, Y. (1990). Heparin uncouples the muscarinic receptors from GK protein in the atrial cell membrane of the guinea-pig heart. Pflügers Archiv 417, 126-128 [Medline]

ITOH T., SEKI, N., SUZUKI, S., ITO, S., KAJIKURI, J. & KURIYAMA, H. (1992). Membrane hyperpolarization inhibits agonist-induced synthesis of inositol 1,4,5-trisphosphate in rabbit mesentric artery. Journal of Physiology 451, 307-328 [Abstract]

JAIMOVICH E., REYES, R., LIBERONA, J. L. & POWELL, J. A. (2000). IP₃ receptors, IP₃ transients, and nucleus-associated Ca²⁺ signals in cultured skeletal muscle. American Journal of Physiology - Cell Physiology 278, C998-1010 [Medline]

KIMURA M., JEANCLOS, E. M., DONNELLY, R. J., LYTTON, J., REEVES, J. P. & AVIV, A. (1999). Physiological and molecular characterization of the Na⁺/Ca²⁺ exchanger in human platelets. American Journal of Physiology 277, H911-917 [Medline]

KISELYOV K., XU, X., MOZHAYEVA, G., KUO, T., PESSAH, I., MIGNERY, G., ZHU, X., BIRNBAUMER, L. & MUALLEM, S. (1998). Functional interaction between InsP₃ receptors and store-operated Htrp3 channels. Nature 396, 478-482 [Medline]

KUNAPULI S. P. & DANIEL, J. L. (1998). P2 receptor subtypes in the cardiovascular system. Biochemical Journal 336, 513-523 [Medline]

MA H. T., PATTERSON, R. L., VAN ROSSUM, D. B., BIRNBAUMER, L., MIKOSHIBA, K. & GILL, D. L. (2000). Requirement of the inositol trisphosphate receptor for activation of store-operated Ca²⁺ channels. Science 287, 1647-1651. [Abstract/Full Text]

MAHAUT-SMITH M. P. (1998). An infra-red light-transmitting aperture controller for use in single cell fluorescence photometry. Journal of Microscopy 191, 60-66 [Medline]

MAHAUT-SMITH M. P., HUSSAIN, J. F. & MASON, M. J. (1999). Depolarization-evoked Ca²⁺ release in a non-excitable cell, the rat megakaryocyte. Journal of Physiology 515, 385-390 [Abstract/Full Text]

MASON M. J., HUSSAIN, J. F. & MAHAUT-SMITH, M. P. (2000). A novel role for membrane potential in the modulation of intracellular Ca²⁺ oscillations in rat megakaryocytes. Journal of Physiology 524, 437-446 [Abstract/Full Text]

MORARU I. I., KAFTAN, E. J., EHRLICH, B. E. & WATRAS, J. (1999). Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP₃ and calcium: simulation of single channel kinetics based on ligand binding and electrophysiological analysis. Journal of General Physiology 113, 837-849 [Medline]

MUSCHOL M., DASGUPTA, B. R. & SALZBERG, B. M. (1999). Caffeine interaction with fluorescent calcium indicator dyes. Biophysical Journal 77, 577-586 [Abstract/Full Text]

PARKER I. & IVORRA, I. (1991). Caffeine inhibits inositol trisphosphate-mediated liberation of intracellular calcium in Xenopus oocytes. Journal of Physiology 443, 229-240

POENIE M. (1990). Alteration of intracellular fura-2 fluorescence by viscosity: a simple correction. Cell Calcium 11, 85-91 [Medline]

RECHES A., ELDOR, A. & SALMON, Y. (1979). The effects of dextran sulfate, heparin and PGE₁ on adenyl cyclase activity and aggregation of human platelets. Thrombosis Research 16, 107-116 [Medline]

RIOS E. & BRUM, G. (1987). Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature 325, 717-720 [Medline]

SAYERS L. G., BROWN, G. R., MICHELL, R. H. & MICHELANGELI, F. (1993). The effects of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-induced calcium release in cerebellar microsomes. Biochemical Journal 289, 883-887 [Medline]

SCHNEIDER M. F. & CHANDLER, W. K. (1973). Voltage dependent charge movement in skeletal muscle: a possible step in excitation-contraction coupling. Nature 242, 244-246 [Medline]

SOMASUNDARAM B. & MAHAUT-SMITH, M. P. (1994). Three cation influx currents activated by purinergic receptor stimulation in rat megakaryocytes. Journal of Physiology 480, 225-231 [Abstract]

TAYLOR C. W. & TRAYNOR, D. (1995). Calcium and inositol trisphosphate receptors. Journal of Membrane Biology 145, 109-118 [Medline]

TERTYSHNIKOVA S. & FEIN, A. (1998). Inhibition of inositol 1,4,5-trisphosphate-induced Ca²⁺ release by cAMP-dependent protein kinase in a living cell. Proceedings of the National Academy of Sciences of the USA 95, 1613-1617 [Abstract/Full Text]

UNEYAMA H., UNEYAMA, C. & AKAIKE, N. (1993). Intracellular mechanisms of cytoplasmic Ca²⁺ oscillation in rat megakaryocyte. Journal of Biological Chemistry 268, 168-174 [Abstract]

VAN HELDEN D. F., IMTIAZ, M. F., NURGALIYEVA, K., VON DER WEID, P. Y. & DOSEN, P. J. (2000). Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus. Journal of Physiology 524, 245-265 [Abstract/Full Text]

WAKUI M., OSIPCHUK, Y. V. & PETERSON, O. H. (1990). Receptor-activated cytoplasmic Ca²⁺ spiking mediated by inositol trisphosphate is due to Ca²⁺-induced Ca²⁺ release. Cell 63, 1025-1032 [Medline]

Acknowledgements

This work was funded by the British Heart Foundation (Basic Science Lectureship, BS 10, to M. P. M.- S.), the Royal Society and the Medical Research Council. The authors wish to thank Jon Holdich for expert technical assistance.

Corresponding author

M. J. Mason: Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK.

Email: mjm39{at}cam.ac.uk

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BEZPROZVANNY I., BEZPROZVANNY, S. & EHRLICH, B. E. (1994). Caffeine-induced inhibition of inositol(1,4,5)-trisphosphate-gated calcium channels from cerebellum. Molecular Biology of the Cell 5, 97-103	[Abstract]
BEZPROZVANNY I. & EHRLICH, B. E. (1994). Inositol (1,4,5)-trisphosphate (InsP₃)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium. Journal of General Physiology 104, 821-856	[Medline]
BEZPROZVANNY I., WATRAS, J. & EHRLICH, B. E. (1991). Bell-shaped calcium-response curves of Ins(1,4,5)P₃ and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 351, 751-754	[Medline]
BOOTMAN M. D., TAYLOR, C. W. & BERRIDGE, M. J. (1992). The thiol reagent, thimerosal, evokes Ca²⁺ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor. Journal of Biological Chemistry 267, 25113-25119	[Abstract]
BOULAY G., BROWN, D. M., QIN, N., JIANG, M., DIETRICH, A., ZHU, M. X., CHEN, Z., BIRNBAUMER, M., MIKOSHIBA, K. & BIRNBAUMER, L. (1999). Modulation of Ca²⁺ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca²⁺ entry. Proceedings of the National Academy of Sciences of the USA 96, 14955-14960	[Abstract/Full Text]
BROWN G. R., SAYERS, L. G., KIRK, C. J., MICHELL, R. H. & MICHELANGELI, F. (1992). The opening of the inositol 1,4,5-trisphosphate-sensitive Ca²⁺ channel in rat cerebellum is inhibited by caffeine. Biochemical Journal 282, 309-312	[Medline]
CUTLER L. S. & CHRISTIAN, C. P. (1984). Inhibition of rat salivary gland adenylate cyclase by glycosaminoglycans and high molecular weight polyanions. Archives of Oral Biology 29, 629-633	[Medline]
DORN W. G. II_. & DAVIS, M. G. (1992). Differential megakaryocytic desensitization to platelet agonists. American Journal of Physiology 263, C864-872	[Medline]
EBERHARD D. A. & HOLZ, R. W. (1988). Intracellular Ca²⁺ activates phospholipase C. Trends in Neurosciences 12, 517-520
FABIATO A. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. American Journal of Physiology 245, C1-14	[Medline]
FINCH E. A., TURNER, T. J. & GOLDIN, S. M. (1991). Calcium as a co-agonist of inositol 1,4,5-trisphosphate-induced calcium release. Science 252, 443-446	[Medline]
GANITKEVICH V. YA. & ISENBERG, G. (1993). Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca²⁺ transients in guinea-pig coronary myocytes. Journal of Physiology 470, 35-44	[Abstract]
GLASS D. B., CHENG, H. C., MENDE-MEULLER, L., REED, J. & WALSH, D. A. (1989). Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein. Journal of Biological Chemistry 264, 8802-8810	[Abstract]
GRYNKIEWICZ G., POENIE, M. & TSIEN, R. Y. (1985). A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450	[Abstract]
HAGAR R. E., BURGSTAHLER, A. D., NATHANSON, M. H. & EHRLICH, B. E. (1998). Type III InsP₃ receptor channel stays open in the presence of increased calcium. Nature 396, 81-84	[Medline]
HILLY M., PIÉTRI-ROUXEL, F., COQUIL, J. F., GUY, M. & MAUGER, J. P. (1993). Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor. Journal of Biological Chemistry 268, 16488-16494	[Abstract]
HINDE A. K., PERCHENET, L., HOBAI, I. A., LEVI, A. J. & HANCOX, J. C. (1999). Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 °C, dialysed internally with cAMP-free and cAMP-containing solutions. Cell Calcium 25, 321-331	[Medline]
IINO M. (1990). Biphasic Ca²⁺ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. Journal of General Physiology 95, 1103-1122	[Medline]
IRVINE R. F. (1990). 'Quantal' Ca²⁺ release and the control of Ca²⁺ entry by inositol phosphates - a possible mechanism. FEBS Letters 263, 5-9	[Medline]
ITO H., TAKIKAWA, R., IGICHI, M., HAMADA, E., SUGIMOTO, T. & KURACHI, Y. (1990). Heparin uncouples the muscarinic receptors from GK protein in the atrial cell membrane of the guinea-pig heart. Pflügers Archiv 417, 126-128	[Medline]
ITOH T., SEKI, N., SUZUKI, S., ITO, S., KAJIKURI, J. & KURIYAMA, H. (1992). Membrane hyperpolarization inhibits agonist-induced synthesis of inositol 1,4,5-trisphosphate in rabbit mesentric artery. Journal of Physiology 451, 307-328	[Abstract]
JAIMOVICH E., REYES, R., LIBERONA, J. L. & POWELL, J. A. (2000). IP₃ receptors, IP₃ transients, and nucleus-associated Ca²⁺ signals in cultured skeletal muscle. American Journal of Physiology - Cell Physiology 278, C998-1010	[Medline]
KIMURA M., JEANCLOS, E. M., DONNELLY, R. J., LYTTON, J., REEVES, J. P. & AVIV, A. (1999). Physiological and molecular characterization of the Na⁺/Ca²⁺ exchanger in human platelets. American Journal of Physiology 277, H911-917	[Medline]
KISELYOV K., XU, X., MOZHAYEVA, G., KUO, T., PESSAH, I., MIGNERY, G., ZHU, X., BIRNBAUMER, L. & MUALLEM, S. (1998). Functional interaction between InsP₃ receptors and store-operated Htrp3 channels. Nature 396, 478-482	[Medline]
KUNAPULI S. P. & DANIEL, J. L. (1998). P2 receptor subtypes in the cardiovascular system. Biochemical Journal 336, 513-523	[Medline]
MA H. T., PATTERSON, R. L., VAN ROSSUM, D. B., BIRNBAUMER, L., MIKOSHIBA, K. & GILL, D. L. (2000). Requirement of the inositol trisphosphate receptor for activation of store-operated Ca²⁺ channels. Science 287, 1647-1651.	[Abstract/Full Text]
MAHAUT-SMITH M. P. (1998). An infra-red light-transmitting aperture controller for use in single cell fluorescence photometry. Journal of Microscopy 191, 60-66	[Medline]
MAHAUT-SMITH M. P., HUSSAIN, J. F. & MASON, M. J. (1999). Depolarization-evoked Ca²⁺ release in a non-excitable cell, the rat megakaryocyte. Journal of Physiology 515, 385-390	[Abstract/Full Text]
MASON M. J., HUSSAIN, J. F. & MAHAUT-SMITH, M. P. (2000). A novel role for membrane potential in the modulation of intracellular Ca²⁺ oscillations in rat megakaryocytes. Journal of Physiology 524, 437-446	[Abstract/Full Text]
MORARU I. I., KAFTAN, E. J., EHRLICH, B. E. & WATRAS, J. (1999). Regulation of type 1 inositol 1,4,5-trisphosphate-gated calcium channels by InsP₃ and calcium: simulation of single channel kinetics based on ligand binding and electrophysiological analysis. Journal of General Physiology 113, 837-849	[Medline]
MUSCHOL M., DASGUPTA, B. R. & SALZBERG, B. M. (1999). Caffeine interaction with fluorescent calcium indicator dyes. Biophysical Journal 77, 577-586	[Abstract/Full Text]
PARKER I. & IVORRA, I. (1991). Caffeine inhibits inositol trisphosphate-mediated liberation of intracellular calcium in Xenopus oocytes. Journal of Physiology 443, 229-240
POENIE M. (1990). Alteration of intracellular fura-2 fluorescence by viscosity: a simple correction. Cell Calcium 11, 85-91	[Medline]
RECHES A., ELDOR, A. & SALMON, Y. (1979). The effects of dextran sulfate, heparin and PGE₁ on adenyl cyclase activity and aggregation of human platelets. Thrombosis Research 16, 107-116	[Medline]
RIOS E. & BRUM, G. (1987). Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature 325, 717-720	[Medline]
SAYERS L. G., BROWN, G. R., MICHELL, R. H. & MICHELANGELI, F. (1993). The effects of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-induced calcium release in cerebellar microsomes. Biochemical Journal 289, 883-887	[Medline]
SCHNEIDER M. F. & CHANDLER, W. K. (1973). Voltage dependent charge movement in skeletal muscle: a possible step in excitation-contraction coupling. Nature 242, 244-246	[Medline]
SOMASUNDARAM B. & MAHAUT-SMITH, M. P. (1994). Three cation influx currents activated by purinergic receptor stimulation in rat megakaryocytes. Journal of Physiology 480, 225-231	[Abstract]
TAYLOR C. W. & TRAYNOR, D. (1995). Calcium and inositol trisphosphate receptors. Journal of Membrane Biology 145, 109-118	[Medline]
TERTYSHNIKOVA S. & FEIN, A. (1998). Inhibition of inositol 1,4,5-trisphosphate-induced Ca²⁺ release by cAMP-dependent protein kinase in a living cell. Proceedings of the National Academy of Sciences of the USA 95, 1613-1617	[Abstract/Full Text]
UNEYAMA H., UNEYAMA, C. & AKAIKE, N. (1993). Intracellular mechanisms of cytoplasmic Ca²⁺ oscillation in rat megakaryocyte. Journal of Biological Chemistry 268, 168-174	[Abstract]
VAN HELDEN D. F., IMTIAZ, M. F., NURGALIYEVA, K., VON DER WEID, P. Y. & DOSEN, P. J. (2000). Role of calcium stores and membrane voltage in the generation of slow wave action potentials in guinea-pig gastric pylorus. Journal of Physiology 524, 245-265	[Abstract/Full Text]
WAKUI M., OSIPCHUK, Y. V. & PETERSON, O. H. (1990). Receptor-activated cytoplasmic Ca²⁺ spiking mediated by inositol trisphosphate is due to Ca²⁺-induced Ca²⁺ release. Cell 63, 1025-1032	[Medline]

				D. Billups, B. Billups, R. A. J. Challiss, and S. R. Nahorski Modulation of Gq-Protein-Coupled Inositol Trisphosphate and Ca2+ Signaling by the Membrane Potential J. Neurosci., September 27, 2006; 26(39): 9983 - 9995. [Abstract] [Full Text] [PDF]

				A. del Valle-Rodriguez, E. Calderon, M. Ruiz, A. Ordonez, J. Lopez-Barneo, and J. Urena Metabotropic Ca2+ channel-induced Ca2+ release and ATP-dependent facilitation of arterial myocyte contraction. PNAS, March 14, 2006; 103(11): 4316 - 4321. [Abstract] [Full Text] [PDF]

				M. S. Imtiaz, C. P. Katnik, D. W. Smith, and D. F. van Helden Role of Voltage-Dependent Modulation of Store Ca2+ Release in Synchronization of Ca2+ Oscillations Biophys. J., January 1, 2006; 90(1): 1 - 23. [Abstract] [Full Text] [PDF]

				J. Martinez-Pinna, I. S. Gurung, C. Vial, C. Leon, C. Gachet, R. J. Evans, and M. P. Mahaut-Smith Direct Voltage Control of Signaling via P2Y1 and Other G{alpha}q-coupled Receptors J. Biol. Chem., January 14, 2005; 280(2): 1490 - 1498. [Abstract] [Full Text] [PDF]

				J. Martinez-Pinna, G. Tolhurst, I. S. Gurung, J. I. Vandenberg, and M. P. Mahaut-Smith Sensitivity limits for voltage control of P2Y receptor-evoked Ca2+ mobilization in the rat megakaryocyte J. Physiol., February 15, 2004; 555(1): 61 - 70. [Abstract] [Full Text] [PDF]

				M. A. Hollywood, G. P. Sergeant, N. G. McHale, and K. D. Thornbury Activation of Ca2+-activated Cl- current by depolarizing steps in rabbit urethral interstitial cells Am J Physiol Cell Physiol, August 1, 2003; 285(2): C327 - C333. [Abstract] [Full Text] [PDF]

				M. P. Mahaut-Smith, D. Thomas, A. B. Higham, J. A. Usher-Smith, J. F. Hussain, J. Martinez-Pinna, J. N. Skepper, and M. J. Mason Properties of the Demarcation Membrane System in Living Rat Megakaryocytes Biophys. J., April 1, 2003; 84(4): 2646 - 2654. [Abstract] [Full Text] [PDF]

				M. S. Imtiaz, D. W. Smith, and D. F. van Helden A Theoretical Model of Slow Wave Regulation Using Voltage-Dependent Synthesis of Inositol 1,4,5-Trisphosphate Biophys. J., October 1, 2002; 83(4): 1877 - 1890. [Abstract] [Full Text] [PDF]

				J. F Risher, H E. Murray, and G. R Prince Organic mercury compounds: human exposure and its relevance to public health Toxicology and Industrial Health, April 1, 2002; 18(3): 109 - 160. [Abstract] [PDF]

				G. D. S. Hirst, N. J. Bramich, N. Teramoto, H. Suzuki, and F. R. Edwards Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl- channels J. Physiol., May 1, 2002; 540(3): 907 - 919. [Abstract] [Full Text] [PDF]