[Ca2+]i oscillations and [Ca2+]i waves in rat megakaryocytes

Cdl cakkJrn (1997) 21(5), 331-344 0 Pearson Professional Ltd 1997

Research

[Ca2+li oscillations and [Ca’+], waves in rat megakaryocytes Svetlana Tertyshnikova*, Alan Fein Department

of Physiology,

University

of Connecticut

Health Center,

Farmington,

Connecticut,

USA

Summary ATP activated [Ca*+], oscillations were measured in single rat megakaryocytes using fluorescence ratio microscopy. With increasing ATP concentration the duration of the [Ca*+], oscillations increased, however, there was considerable variation from cell to cell in the absolute value of the peak [Ca*+], and the frequency and duration of the oscillations. This variation depended, in part, on the level of Fura- loading suggesting that megakaryocytes are sensitive to buffering of [Ca2+li by Fura-2. Agents, that increase the level of intracellular cGMP (sodium nitroprusside and 8-pCPT-cGMP) or CAMP (prostacyclin, IBMX, forskolin and 8-bromo-CAMP) inhibited [Ca*+], oscillations. Despite the large cell to cell variation in the patterns of [Ca*+], oscillations, reapplication of the agents that elevated CAMP or cGMP inhibited the oscillations similarly. Using video rate fluorescence ratio imaging we found that the agonist-induced [Ca*+], oscillations were the result of a well-defined [Ca*+],wave, which spread across the cell with an average speed of about 35 pm/s, during the rising phase of each oscillatory spike. After reaching a peak, [Ca*+], decreased uniformly across the whole cell during the falling phase of the spike. Analysis of the temperature dependence of [Ca*+], waves showed that the rate of [Ca*+], decay exhibited a strong temperature dependence (a,, -4), whereas, the rate of rise exhibited a weak temperature dependence (Cl,, -1.3), suggesting, that the rate limiting process for [Ca*+], wave propagation in rat megakaryocytes is the rate of [Ca*+],diffusion.

INTRODUCTION

Platelets play an important role in hemostasis and cardiovascular diseases, and are one of the cell types, where [Caz+], regulation is currently under intense investigation. Increases in cytosolic free calcium, [Ca2+li, have been shown to play a central pivotal role in platelet activation (see reviews by [l-4]). For example, an increase of [Ca2+li resulting from exposure to Ca2+ ionophore will trigger platelet aggregation and secretion [5]. Most

Received Revised Accepted

29 October

1996

17 January 1996 17 January 1997

platelet activators elevate [Ca’+], by an inositol 1,4,5t&phosphate mediated release of Ca2+ from intracellular stores, as well as stimulation of the entry of extracellular Ca2+ [ 1,6-81. In contrast to platelet activators, platelet inhibitors elevate the level of CAMP or cGMP and, thereby, antagonize the activator evoked [Ca’+], elevation [1,3,8,9]. Many, but not all, of the effects of CAMP and cGMP are thought to be mediated by CAMP- and cGMPdependent protein kinases, respectively [2,10,1 11. However, the precise mechanism by which elevated CAMP and cGMP inhibits platelet activation has yet to be determined. Since platelets are small and fragile, there have been only a few studies of [CaZ+],regulation in platelets at the single cell level [ 12-151. Some researches have used

Correspondence to: Dr Alan Fein, Department. of Physiology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 060303505, USA Tel: +1 860 679 2263: Fax: +I860 679 1269 E-mail: [email protected]

Abbreviations used: [Caz+],, cytosolic free Ca*+ concentration; IKcB,Ca2+activated K+-current; ATP, adenosine 5’-triphosphate; PGI,, prostacyclin; IBMX, 3-isobutyl-1-methylxanthine; 8-bromo-CAMP, 8-bromoadenosine 3’-5’-

‘Permanent address: Academy of Science,

cyclic monophosphate; 8-pCPT-cGMP, 8-(4-chlorophenylthio)guanosine 3’,5’-cyclic monophosphate; SNP, sodium nitroprusside.

Branch of Institute of Bioorganic Pushchino, Russia

Chemistry,

Russian

331

332

S Tertyshnikova, A Fein

megakaryocytes, instead of platelets, taking advantage of their large size and the fact that megakaryocytes, as the progenitors of platelets, share many functional properties with platelets, including responsiveness to various bioactive substances, which promote or inhibit platelet activation [ 16-201. [Ca2+li oscillations occur in both excitable and nonexcitable cells and are thought to be an important feature of cell function (for reviews, see [21,22]). It is only recently that it has become apparent for platelets, as it has for many other cell types, that activator induced Ca2+release results in an [Ca2+li oscillation [ 12-14,231. Likewise, in megakaryocytes recent studies have shown that activator-induced release of calcium from intracellular pools [24] also results in an calcium activated potassium current (I,d oscillation in rat megakaryocytes, which reflects an underlying [Ca2+], oscillation [ 16,171. Because [Ca2+], oscillations have only recently been discovered in platelets and megakaryocytes, very little is known about the mechanisms underlying these oscillations. In the present study we have used fluorescence ratio microscopy of Fura- loaded rat megakaryocytes to measure directly agonist-induced [Ca2+], oscillations at the single cell level. We have shown that agents which elevate intracellular CAMP and cGMP reversibly inhibit these agonist-induced [Ca2+li oscillations. Using video rate fluorescence ratio imaging microscopy, we have found that [Ca2+], oscillations in rat megakaryocytes are the result of [Ca2+li waves and we have characterized these [Ca2+li waves. MATERIALS

AND

METHODS

Preparation of rat megakaryocytes

Adult male Wistar rats weighing 150-250 g were anaesthetized by inhalation of an overdose of isoflurane and then sacrificed by decapitation. The tibial and femoral bones were isolated and an incision was made on the end of each bone, then bone marrow was washed out with standard external solution (see below) by the use of a disposable syringe and needle. After filtration through a 75 pm nylon mesh to eliminate large masses of cells, the suspension was spun and washed twice before storage in standard external solution. The standard external solution contained NaCl 140 mM, KC1 5 n&I, MgCl, 1 mM, CaCl, 2 mM, glucose 10 mM, HEPES 10 mM, adjusted to pH 74 with NaOH, supplemented by 0.1% bovine serum albumin @ISA). Loading cells with Fura-S/AM

For intracellular loading of Fura-2, cells were resuspended in standard external solution, containing 2.5 @I Cell Calcium

(1997)

21(5),

331-344

Fura-2/AM and 0.01% Pluronic F-127, transferred onto glass coverslips and incubated for 30 min, the coverslips with adherent cells were then washed several times with the standard external solution, and kept in the dark at room temperature until use. Fura-2/AM, dissolved in dimethylsulfoxide (DMSO) and the surfactant (Pluronic F127) were well mixed together before being added to the standard external solution containing the cell suspension. Materials

Fura- acetoxymethyl ester (Fura-2/AM) and Pluronic F127 were obtained from Molecular Probes Inc. (Eugene, OR, USA). 8-(4chlorophenykhio)guanosine 3’,5’-cyclic monophosphate (8-pCPT-cGMP) was from BioLog (La Jolla, CA, USA). All other agents, were purchased from Sigma Chemical Co. (St Louis, MO, USA). Agonist application

Activators and inhibitors (ATP, or the mixture of ATP with any one of the following: PGI,, IBMX, forskolin, 8bromo-CAMP, SNP or %pCPT-cGMP), were dissolved in the standard external solution and applied directly to single megakaryocytes using a DAD-6 computer controlled local superfusion system (Adams & List Assoc., Ltd, New York, NY, USA). The local superfusion system allowed us to apply agents rapidly to individual cells from up to 6 reservoirs connected to a micromanifold. The output tube of the micromanifold (100 pm inside diameter) was placed approximately within 300 un~ of the cell, the puff pressure was adjusted to achieve rapid agonist application while avoiding any mechanical disturbance of the cell. The time delay for arrival of agonists at the cell was measured and accounted for in the figures. Measurement of [Ca*+],

Coverslips with attached Fura- loaded megakaryocytes formed the bottom of the teflon recording chamber (LUCSD chamber, Medical Systems Corp., Greenvale, NY, USA) which was filled with 1 ml of standard external solution. The chamber was placed on the stage of an inverted microscope (Nikon, Diaphot TMD) and viewed with either an Nikon Fluor 40x 1.3 NA or Nikon Fluor 100x 1.3 NA oil immersion lens. Megakaryocytes were clearly distinguished from other bone marrow cells on the basis of their large size (25-50 i.un) and multilobular nucleus [ 171. Recordings were made at room temperature within 6 h of isolation. The microscope sits on a floating table (TMC, Peabody, MA, USA) for vibration isolation from the environment and is surrounded by a darkened Faraday cage to eliminate electrical interference and block out stray light. 0 Pearson

Professional

Lfd 1997

[Ca2’1, oscillations

Whole cell fluorometry was accomplished using an Ionoptix photon counting fluorescence subsystem (Ionoptix, Milton, MA, USA). Light was delivered to the epifluorescence port from a chopper based dual excitation 75 watt xenon arc light source via a liquid light guide. Light emerging from the light guide strikes a dichroic mirror that reflects light below 430 nm to the microscope objective which focuses the light onto the cell. Fluorescence emission is then collected by the same objective and passes through the dichroic mirror and then through a 5 10 nm wide band barrier filter before reaching the photon counting photomultiplier. The microscope adapter which holds the photomultiplier has a cell framing aperture associated with it, so that a single cell can be framed and measured. The two fluorescence intensities and their ratio were accumulated and transferred through an interface to a personal computer (Gateway Pentium, 133 Mhz with 64 MB RAM). The data acquisition was done on-line using the PIA program (IonOptix, Milton, MA, USA). The time resolution was set at 0.1 s by averaging 3 points to obtain a better signal-to-noise ratio. Background fluorescence was measured and subtracted at the end of every experiment. [Ca2+liwas calculated from the ratio of 340/380 mn fluorescence values (after subtraction of background fluorescence) as described by Grynkiewicz et al. [25]. The values for R&, R-, and the constant Sf,/Sb, were calculated from measurements with 2 l.tIvl Fura- free acid in solution in vitro. The dissociation constant for the Fura-2*Ca2+ complex was taken as 225 nM (Molecular Probes). Video rate fluorescence ratio imaging was performed on the same Nikon, Diaphot microscope using the Nikon Fluor 100x 1.3 NA oil immersion lens, and the IonOptix dual-wavelength excitation source and interface, but the photomultiplier assembly was replaced with an intensified CCD video camera (IonOptix) as the imaging device. The two fluorescence images and their ratio were digitized at &bit resolution and stored directly in the RAM of the personal computer. The ratio images were converted to [Ca*+], and expressed in gray scale. The data acquisition and preliminary analysis were done on-line using the IonWizard software (IonOptix). Images pairs were obtained at 0.033 s intervals and 3 frames were averaged to obtain a better signal-to-noise ratio. Background was measured for a cell free region and subtracted from the Fura- Ca2+ signal for the cell at the end of the experiment. For this microscope and image processing system, 1 pixel = 0.33 pm. [Ca”], waves were identified by reviewing data played back frame by frame in a movie type mode. When ratioing, pixels with an intensity below a threshold value are rejected (that is, the corresponding point on the ratio image is displayed as black). Because the 380 image dims when [Ca2+lirises, some pixels at the periphery of the cells fall below the threshold value: this 0 Pearson

Professional

Ltd 1997

and [C&J

waves in rat megakatyocytes

333

gives the impression that the cells are shrinking on stimulation (seefor example Fig. i’C-2b). Quantitative analysis of the speed of [Ca’+], wave propagation was done using the program NIH Image. For quantitative analysis, a rectangular segment was overlaid on the cell with its long axis oriented in the direction of wave propagation (seeFig. 6B). For this rectangular selection the plot profile was produced as a ‘column average plot; where the width of the plot was equal to the larger dimension of the rectangular area and each point in the plot represents the average gray value of the 20 pixels in the column across the smaller dimension of the rectangular area (Pig. 6B). To determine the speed of the wave as it crossed the cell, the fluorescence profile across the rectangular area was measured at two time points 0.1 s apart and the distance between the points in the cell where the fluorescence was 50% of the peak A[Ca2+liwas determined, and the velocity was calculated as V = Ax/At. For determining the wave front position at each time point, locations in the cell where [Ca’+], was between 40% and 60% of the peak of A[Ca2+liwere plotted on the graph, where the abscissa represents time and the ordinate represents location along the selected rectangular segment (Pig. 6C). The average speed of the [Caz+], wave was measured by taking the slope of the wave front position graph. For determination of the temperature dependence of the kinetics of the rising and falling phases of the [Ca’+], waves in megakaryocytes, the temperature of the external solution was varied over the range 24- 14°C. The bath temperature was rapidly cooled to the low temperature point by addition of chilled external solution. The miniature probe of a digital thermometer was placed in close proximity to the cell to monitor temperature. [Ca’+], oscillations made up of individual [Ca2+],waves, evoked by application of 100 @I ATP, were recorded as the temperature increased from a low point to room temperature over about 3-5 min. Reapplication of cold solution affected the [Ca’+], oscillations similarly. Curve fitting was done using SigmaPlot for Windows (Jandel Scientific, San Rafael, CA, USA). RESULTS

ATP-induced [Caz+],oscillations megakaryocytes

in single rat

After a short latent period, following application of ATP, [Ca*+], began to oscillate reaching peak values in the range of about 300-1000 nM during the individual spikes that made up the oscillation. initially the oscillation was relatively fixed in frequency and amplitude (Fig. IA-C) however, with time the interval between [Ca*+], spikes increased and eventually the [Ca’+], oscillation ceased. The peak [Ca2+li level, achieved in the individual spikes that made up the oscillation, was independent of Cell Calcium (1997)

21(5),

331-344

334

S Tertyshnikova,

A Fein

A

zc

900

J/

k

22 80 ES0 -

OL 0 100

A;2 -

time, s

Ii

$40 Y 2 yj20 & 2

o-,

time, s

Ii

8 40 Y B $20 0O

oeazEooo

t $0-t

I

0 00

&e O

0 100 200 300 dye load (F 380 nm), countsIms

c’

0 0

0

0 o” I ti-

8

0

100 200 300 dye load (F 380 nm), counts/ms

500 c

: 3 80

5A; o_ 0 0

100

200

300 400

500 600

D

time, s

c

700

0 0 00

00 00 0 O ooo” 0 0 0 0

1

Q 100

200

300

400

10 100 ATf’ (PM)

dye load (F 380 nm), counts/ms

Fig. 1 Concentration-dependence of ATP-induced [Ca2+],osciiiations in single rat megakaryocytes. (A-C) Show the relationship between the number of spikes in the [Ca*+], oscillation, for a constant ATP concentration, and the ceil dye load, expressed arbitrarily as the fluorescence intensity in units of countsIms, for the 380 nm wavelength signal (F 380 nm). Each data point was obtained from an individual cell. The concentrations of ATP used were 10 (A) (n = 18 ceils), 50 (B) (n = 28 cells) and 100 yM (C) (n = 24 ceils). The inserts show a typical example of a cellular response at each concentration with the time of ATP addition indicated by an arrow in each trace. (D) Concentration dependence of the number of spikes induced by ATP (mean f SEM). Only cells with fluorescence intensity between 70 and 150 (i.e. ‘ceils lightly loaded with Fura-2’) (see Results) were included in the graph.

the ATP concentration. We observed considerable intercellular variation in the absolute value of the peak [Ca*+], concentration achieved during individual spikes but, for each individual cell, the peak-to-peak variation and the variation from one ATP application to the next was not as great. It is apparent, from the plots and representative single cell responses to varying ATP concentrations (Fig. 1-C) that the duration of the [Caz+li oscillations, and thereby the total number of spikes, increased with increasing ATP concentration. Cell Calcium

(1997)

21(5),

331-344

It can be seen in Figure lA-C that there is considerable cell-to-cell variation in the number of [Caz+li spikes elicited at each ATP concentration. At the higher ATP concentrations, the total number of [Caz+], spikes varied by about an order of magnitude. This variability was partially dependent on the amount of dye loaded into the cells (Fig. lA-C). It was possible to assess the amount of dye loaded into the cells by comparing the resting levels of 380 mn fluorescence because most of the cells had a similar resting [Ca2+lilevel between 80 and 130 r&I. The 0 Pearson Professional Ltd 1997

[C$+J oscillations and [C&J waves in rat megakaryocytes

data in the plots of Figure lA-C show that, as the amount of dye loaded into megakaryocytes increases, the number of [Ca*+], spikes elicited upon exposure to ATP decreases. In order to maximize the number of [Caz+], spikes elicited upon exposure to ATP in our further studies, we only studied cells with an 380 mn fluorescence intensity between 70 and 150 (i.e. ‘cells lightly loaded with Fura-2’). Additionally, there was some variability in the [Ca2+liresponses of cells having similar 380 nm fluorescence intensity, which might be explained by heterogeneity in the sensitivity of individual cells to ATP [ 16,261, and also by differing cell sensitivity to [Ca2+li buffering by Fura-2. The graph in Figure lD, shows the average number of [Ca2+lispikes elicited by different concentrations of ATP for cells lightly loaded with Fura-2. Maximal responses were observed with 100 i.tM ATP, the largest concentration used. Therefore, in our ensuing experiments, we studied [Ca2+li oscillations in ‘lightly loaded’ cells exposed to 100 uM ATP. Regulation of [Ca2+], oscillations intracellular CAMP and cGMP

by agents that elevate

It has been shown that agents such as endothelium derived relaxing factor (EDRF), PGI,, SNP, and cell permeant PGeM

A

ATP IOOpM

forskolin 0.5 PM

8komo-cAMP

D

335

analogs of CAMP and cGMP inhibit calcium mobilization from intracellular stores in platelets [ 1, lo]. In rat megakaryocytes, IBMX reversibly and forskolin irreversibly inhibited ATP-induced I,, oscillations [ 16,171. We wanted to determine whether agents that elevate CAMP and cGMP reversibly inhibited ATP-induced [Ca2+], oscillations in rat megakaryocytes at concentrations comparable to those at which these agents inhibited calcium mobilization in platelets and IKCaoscillations in rat megakaryocytes. As can be seen in Figure 2A, 2 @4 prostacyclin [271, which is known to activate platelet adenylate cyclase [28] and thereby raise CAMP levels, reversibly inhibited the [Ca2+li oscillation induced by 100 nIvl ATP. Likewise, 5 @I of the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) [8] (Fig. 2B), which raises CAMP levels by inhibiting CAMP degradation via phosphodiesterase [29], reversibly inhibited the ATP-induced [Ca2+], oscillations. We also found that forskolin (0.5 r-LM) [30,31] (Fig. 2C), which elevates CAMP by bypassing the receptor and directly activating adenylate cyclase, reversibly inhibited the [Ca’+], oscillation induced by ATP. Lastly, 250 l,tIvl of the hydrolysis resistant CAMP analog (8-bromo-CAMP) inhibited the ATP-induced [Ca2+lioscillation (Fig. 2D). The suppression of the ATP-induced [Ca2+], oscillation by

B

IBMX 5pM

-

ATP IOOpM

ATP 100 uM

25OpM

ATP IOOpM

Fig. 2

Agents, that elevate intracellular CAMP, reversibly inhibit [Ca’+], oscillations in single rat megakaryocytes. (A) PGI, (prostacyclin), (B) IBMX, (C) forskolin and (D) 8-bromo-CAMP inhibit ATP-induced [Caz+li oscillations in rat megakaryocytes. Each agent was applied during the period, indicated by the horizontal bars. Each curve is typical of the data obtained from 7-12 similar experiments.

Q Pearson Professional Ltd 1997

Cell Calcium (1997) 21(5), 33 l-344

336

S Tertyshnikova,

A Fein

SNP IOOpM

A

ATP 1 OOpM

have a similar mechanism of inhibition induced [Ca2+],mobilization.

of activator-

[Ca*+], waves in rat megakaryocytes

8-pCPT-cGMP 0.5 mM ATP 100 fl

B

f 8L 50 s Fig. 3 Aoents, that elevate intracellular cGMP reversiblv inhibit [Ca2+li o&llations in single rat megakar-yocytes. (A) SNP (sodium nitroorusside) and (B) 8-oCPT-cGMP inhibit ATP-induced fCa*+l. oscillations in single rat megakaryocytes. Each agent was’appl$d during the period, indicated by the horizontal bars. Each curve is typical of the data obtained from 4-7 similar experiments.

prostacyclin, IBMX, forskolin and 8-bromo-CAMP suggests that activation of adenylate cyclase and the resulting rise in CAMP reversibly inhibits the [Ca’+], oscillations in megakaryocytes. In our experiments, agents which elevate cGMP also reversibly inhibited the ATP-induced [Ca2+],oscillations in megakaryocytes (Pig. 3A,B). The nitrovasodilator sodium nitroprusside (SNP), a nitric oxide donor which raises cGMP levels by activating soluble guanylate cyclase [27,32], reversibly inhibited the [Ca2+lioscillations at a concentration of 100 @I (Fig. 3A). We also found that the hydrolysis resistant cGMP analog S-(4-chlorophenylthio)guanosine 3’,5’-cyclic monophosphate (8-pCP’I-cGMP) at a concentration of 250 @I reversibly inhibited the ATPinduced [Ca*+], oscillations (Fig. 3B) [32]. The suppression of the ATP-induced [Ca2+], oscillations by SNP and 8pCPT-cGMP suggests that activation of guanylate cyclase and the resulting rise in cGMP reversibly inhibit the oscillation. Taken together, the results shown in Figures 2 and 3 indicate that agents which elevate CAMP and cGMP, reversibly inhibit [Ca’+], oscillations in rat megakaryocytes, suggesting that megakaryocytes and platelets Cell Calcium (1997) 21(5), 331-344

In many cell types, agonist-induced [Ca2+lirelease often takes the form of a wave, that spreads to every region of the cell (for review, see [21,22,33]). We used video rate fluorescence ratio imaging microscopy to study the spatial distribution of [Ca2+], in single rat megakaryocyte during [Ca2+lioscillations (images were taken every 0.033 s and 3 frames were averaged). We found that ATP-induced [Ca2+], oscillations in rat megakaryocytes were the result of [Ca2+],waves. The increase in [Ca2+], during the rising phases of the [Ca2+li spikes that made up the [Ca2+], oscillations was observed to be the result of a well defined wave, that started locally and then propagated across the cell. An example of such a [Ca2+liwave is shown in Figure 4. The cell was stimulated by application of 100 @I ATP, which elicited a series of [Ca2+li waves, one of which is shown starting at the upper region of the cell body that then spread to the lower region of the cell. After reaching a peak (Fig. 4, frame 19.3 s), [Caz+], decreased uniformly across the whole cell, during the falling phase of the spike. Similar [Ca2+],waves were observed in 20 cells examined by video rate fluorescence ratio imaging. In 17 of these cells, each [Ca2+],wave began at the same location in the cell. In the other 3 cells the site of initiation and hence the direction of propagation of individual waves changed during an [Ca2+], oscillation. In one cell (shown in Fig. 5) the point of initiation and direction of propagation of individual waves reversed direction from spike to spike during the [Ca2+],oscillation. Not shown in Figure 5 is the uniform [Ca2+], decay during the falling phase of each spike of the oscillation. Similar results to those in Figure 5 were observed in 2 other cells, but the direction of propagation only reversed for one spike of the [Ca’+], oscillations in the two cells. These fmdings suggest that rat megakaryocytes are not functionally polarized for the initiation of [Ca2+liwaves. The method used to analyze the velocity of [Ca2+li waves is illustrated in Figure 6. At different time points, the increase in [Ca2+li, A[Ca2+], was averaged across a 20 pixels wide region of interest indicated by the rectangular selection superimposed on the cell image (Fig. 6B), and plotted against distance that is the length of selection (25 l.trn for this cell) (see Fig. 6B, and also Materials and methods). A wave was defined as passing a point when A[Ca2+ji reached 50% of its maximum value at that point (Fig. 6B). The wave front position at each time point was plotted against time (seeMaterials and methods) and the velocity was estimated by fitting the data with a straight line the slope of which gave the speed of the 0 Pearson Professional Ltd 7997

[C$+J oscillations

and [C&J waves in rat megakatyocytes

337

A

16

19

20

21

22 time, s

B

18.3 s

18.4 s

18.5 s

18.6 s

18.7 s

18.8 s

18.9 s

19.0 s

19.1 s

19.3 s

19.5 s

19.7 s

Fig. 4 ATP-induced [Caz+].oscillations in rat megakaryocytes loaded with Fura- are the result of [Ca*+],waves. (A) [Caz+li spike on an expanded time scale. (B) kequence of selected images of [Ca’+], for this spike. Each image in (B) is an average of 3 consecutive frames. The rise of [Ca’+], started in the upper region of the cell, spread across the cell as a well-defined wave and, after reaching a peak, decreased uniformly during the falling phase of the spike. In these images, [Ca*+], is represented in gray scale, according to the scale at the bottom of the figure.

wave (Fig. 6C). Such measurements yielded speeds for the [Caz+], waves which ranged from 26 to 48 pm/s (33.6 + 6.2 pm/s, mean 5 SD, for 16 waves in 6 cells). Since the wave spread across the cell during the rising phase of the [Ca*+], spike, we also estimated the speed of the wave by dividing the cell diameter by the time it takes for [Ca2+li to go from 10% to 90% of its maximum amplitude 1341. The wave velocity obtained from the same 6 cells simply by dividing the cell diameter by the time it takes for [Ca2+lito go from 10% to 90% of its maximum amplitude gave a value of 38.0 f 2.6 pm/s (mean * SD), indicating that either method gives approximately the same value for the [Ca2+],wave propagation velocity. Temperature dependence of [Caz+], wave propagation rat megakaryocytes

in

One approach that might prove useful in the determination of the molecular mechanisms involved in agonistinduced [Ca2+], oscillations in rat megakaryocytes would be to examine the temperature dependence of [Ca2+], wave propagation 1351. If diffusion of a molecule such as 0 Pearson Professional L td 1997

Ca2+ was rate limiting for the propagation of the [Ca2+li wave, then the speed of propagation would be expected to be characterized by a relatively low temperature dependence with a Q,, value less then 2.0 [35-371. If, on the other hand, the speed of [Ca2+], wave propagation were strongly temperature dependent, then a nondiffusional process would be suggested [37-391. To estimate the temperature dependence of the speed of [Ca2+li wave propagation and the kinetics of [Ca2+li spike decay, the rising phase of the spike was fit by a straight line and a single exponential plus a constant was used to fit the falling phase of the spike. The fit was carried out for values of the [Ca2+], spike between 10% and 90% of its maximum amplitude (n = 6 cells) (Fig. 7A). We assume that the time it takes for [Ca’+], to go from 10% to 90% of its maximum amplitude is the approximate time it takes for the wave to propagate across the cell (see above). The rate of rise and the decay rate were normalized to their values at room temperature, so they could be placed on the same graph (Fig. 7s). Normalizing the rate of rise also has the advantage that the cell diameter drops out of the calculation when going from rate of rise to propagation Cell Calcium (1997) 21(5), 33 l-344

338

S Tertyshnikova,

A Fein

velocity. The slope of the straight line used to fit the rate of rise data gives a QIO- 1.3 while the slope of the line used to fit the decay rate data gives a QIO -4.1 (Fig. 7I3). It can be seen in the images in Figure 7C-la and 7C-lb, that the rate of [Ca*+], wave propagation was not significantly altered when the bath temperature went from 22°C to 17°C directly confirming that the speed of the [Caz+li

wave propagation in megakaryocytes has a weak temperature dependence (Fig. 7EI).Similar results to those in Figure X-la and lb were obtained by using video rate fluorescence ratio imaging in 2 other cells. In contrast, the images in Figure 7C-2a and 7C-2b show that the rate of [Caz+], decay during the falling phase of the spike was significantly prolonged when the

0.6 0.4

f 0.6 2 0.4 uj0.2

0.2

0.0 0

20

40

60

80

time, s

11.6s

11.8 s

12.0 s

12.2 s

18.5 s

18.7 s

18.8s

19.0 s

19.1 s

29.6 s

29.7 s

29.8 s

29.9 s

30.1 s

5

6 63.9 s

Fig. 5 [Ca’+], waves in rat megakaryocytes can change direction during a [Ca2+li oscillation. The top trace shows a sequence of six spikes, labeled 1 to 6 during an ATP (100 FM) induced [Ca2+],oscillation. Each sequence of images, labeled 1 to 6, corresponds to the rising phase of each spike in the oscillation. In these images, [Ca*+], is represented by a gray scale according to the scale above the images. Note that the cell moved slightly during the experiment.

Cell Calcium (1997)

21(5),

331-344

0 Pearson Professional Ltd 1997

[Ca2’1, oscillations and [C@ji waves in rat megakaryocytes

20

339

Cim

C / ,Yf 0

pm

, ‘I 18.4

18.8

18.8

19.0

time, s Fig. 6 Determination of wavefront velocity during a [Ca2+li wave. (A) Graph shows [Ca2+],profiles for two consecutive images (see Fig. 4, frames 18.7 s and 16.8 s). [Ca*+], profiles were obtained for the rectangle in (B), where the length of the rectangle is 25 urn and each point in the plot represents the average [Ca*+], value of the 20 pixels across the width of the rectangle. The plot profile curves were well fit by Boltzmann’s equation, indicating that the wave front propagation is compatible with diffusion. (C) Points within the rectangle for which 0.25 uM < [Ca2+], c 0.35 uM are plotted against time. The data were fit by a linear regression, the slope of which reflects the rate of wave front velocity. The average wave front velocity for this cell was 35 pm/s.

bath temperature was lowered from 22°C to 17”C, confirming the data in Figure 7B. DISCUSSION

Using Fura- as a calcium indicator, we have demonstrated that rat megakaryocytes exhibit an ATP induced [Ca’+], oscillation. The duration of the oscillation is dependent on the concentration of ATP, although at all concentrations the oscillations cease with prolonged application of ATP. The cessation of the oscillations could be due to desensitization of the ATP receptor [ 161. Using the nystatin-perforated patch-clamp technique, Uneyama et al. characterized Ca2+-activated K+-current (I& oscillations in rat megakaryocytes, in response to ATP [ 16,171. The frequency of these IKCaoscillations are considerably higher than agonist-induced [Ca2+], oscillations in other nonexcitable cells, such as hepatocytes [26], fibroblasts [40], endothelial cells 1411and pancreatic acinar cells 1421. In our experiments, the frequency of [Ca2+li oscillations was 2-6 spikes/mm at 100 uIVI ATP, which is similar to frequencies reported for other nonexcitable cells, which were studied by direct monitoring of [Ca2+], in Fura- loaded cells and considerably slower 0 Pearson

Professional

Ltd 1997

than what was reported for the I,, oscillations in megakaryocytes (lo-12 per min at 10 @I ATP) [16,17]. It has been shown in Fura-Zloaded hepatocytes by additional loading with Fura-2/AM, that increasing the buffering capacity by increasing the Furaload markedly decreased the frequency of the oscillatory response elicited by phenylephrine 1261.Likewise, loading rat megakaryocytes with the calcium buffer BAPTA by incubating the cells with BAPTA/AM for 30 min at concentrations between 0.1-10 @v¶disrupted the Lti oscillations elicited by 10 PM ATP [ 161.At 1.O l.tM BAPTA/AM, all but the first spike was inhibited, and at 10 pM BAPTiVAM IKcaoscillations were completely blocked. Similarly, in our experiments 10 @I ATP elicited 1 or 2 transient [Ca2+], spikes (Fig. 1A). We found that the ATP concentration (50-100 PM ATP) required to induce long-lasting [Ca2+li oscillations in Fura- loaded rat megakaryocytes (seeFig. 1) was much higher than the ATP concentrations reported to induce IKCaoscillations in rat megakaryocytes [ 161. Taken together, these findings suggest that increasing the buffering capacity for Ca2+with buffers such as BAPTA or Fura- significantly inhibits [Ca2+], oscillations in rat megakaryocytes. This is the most likely reason why previous reports concerning agonist-induced [Ca*+], responses Cell Calcium

(1997)

21(5),

331-344

340

S Tertyshnikova, A Fein

m 0

decay rate rate of rise

0.2 J, ----p-T--T----T--T-l 13 15 17 19 temperature

C

21 (OC)

23

25

la

lb

2a 86.6

86.0

57.0

87.2

07.4

87.5

87.0

BB.0

2b

Fig. 7 Temperature-dependence of [Ca2+],spikes in rat megakaryocyte. (A) Superimposition of two [CaZ+],spikes recorded at 22°C and 17°C from the same cell. The rising phase was ftt by a straight line the slope of which was taken as the rate constant for the rising phase. The falling phase was well fit by a single exponential plus a constant be* + a, the coefficient k was taken as the rate constant for the falling phase. (B) The data for the rate of rise and the decay rate were normalized to their values at room temperature, which was between 22°C and 24°C (n = 6 cells). The slope of the straight line that was fit to the rate of rise gave a Q,,= 1.3 and that for the decay rate gave a Cl,,= 4.1. (C) Sequence of selected images of [Caz+li for the two [Caz+li spikes in (A). la - rising phase at 22°C; 1b - rising phase at 17%; 2a falling phase at 22°C; 2b -falling phase at 17°C. Because the 380 image dims when [Ca’+], rises, some pixels at the periphery of the cell fall below the threshold value: this gives the impression that the cell is shrinking at the peak of the [Caz*li wave.

of megakaryocytes using image analysis or simple fluorometry of Fura- loaded cells failed to detect the oscillatory responses reported here [ 1%20,241. Although increasing the Ca*+ buffering capacity of rat megakaryocytes inhibited the induction of [Caz+],oscillations by ATP, the ability to inhibit these oscillations by agents that elevate CAMP and cGMP (Pigs 2 & 3, respectively) appeared to be unaffected. We found that IBMX Cell Calcium

(1997)

21(5),

331-344

and forskolin inhibited [Caz+li oscillations at the same concentrations in Fura- loaded rat megakaryocytes (Fig. 2) as they inhibited I,, oscillations in these same cells [ 171. In our experiments, forskolin reversibly inhibited the ATP induced [Ca2+],oscillations whereas the effect of forskolin on I,, oscillations appeared to be irreversible. This difference might possibly be explained by a direct inhibitory effect of forskolin on the calcium activated

0 Pearson

Professional

Ltd 1997

[C&ji oscillations and [GY?+]~ waves in rat megakaryocytes

potassium channels that give rise to I,,. In other cell types forskolin has been shown to directly inhibit voltage gated potassium channels [43-46]. The other agents that elevate cAMP (prostacyclin and 8-bromo-CAMP) and cGMP (SNP and 8-pCPT-cGMP) inhibited [Ca2+li oscillations in Fura- loaded rat megakaryocytes at concentrations similar to those at which they inhibit Caz+mobilization in platelets (seeResults). Mechanisms of [Ca’+], oscillations

Many non-excitable cells exhibit [Caz+li oscillations and waves when stimulated with agonists [21,22]. Although a number of models have been proposed (for review, see [21,22,33,47], there is no general consensus about the underlying cellular mechanisms responsible for [Ca2+li oscillations and wave propagation. It seems likely that actual mechanisms may differ from one cell type to another. All the models for non-excitable cells agree on the initial phase of the process: agonist stimulation of a receptor leads to the activation of phospholipase C and the breakdown of phosphatidylinositol4,5-bisphosphate into two important second messengers, inositol 1,4,5trisphosphate (IP,) and diacylglycerol[47]. Diacylglycerol activates protein kinase C [48], while IP, binds to a receptor that acts as a channel in the membrane of an IP,-sensitive Ca2+store (which is believed to be the endoplasmic reticulum) and releases Ca2+ into the cytoplasm [49]. In most non-excitable cells it is possible to elicit an abbreviated [Ca2+li oscillations in the absence of extracellular Ca2+ [16,50-531. This suggests that the source of Ca2+for the oscillation is an intracellular store, but that influx of Ca2+across the plasma membrane is normally required to replenish the intracellular stores with Ca2+. Therefore, most models have concentrated on mechanisms for the oscillatory release of [Ca2+] from one or more intracellular stores and can be distinguished by whether IP, concentrations oscillate as well as [Ca2+], and whether [Ca2+li stimulates or inhibits further release of Ca2+ from intracellular stores. One group of models postulate that the IP, concentration oscillates, these include: (i) the agonist-receptor oscillator model [54-571; and (ii) the IP3-Ca2+ cross-coupling model of Meyer and Stryer 158,591. The Meyer and Stryer model has been found to be most consistent with the properties of [Caz+], oscillations in fibroblasts 1401. Neither of these models appears to apply to rat megakaryocytes because it has been found that internal dialysis of IP, from a patch pipette causes IKC.oscillations in individual rat megakaryocytes [ 171, indicating that [Ca2+], oscillations occur in the presence of a constant concentration of IP,. Several models which do not require oscillations in IP, to induce the oscillations in [Ca2+li have been proposed. 0 Pearson

Professional

Lfd 1997

341

The Ca2+-induced Ca2+release model [60,6 I] invokes two types of Ca2+pools and proposes that Ca2+is first released from an IP,-sensitive pool and, in the continued presence of IP,, the released Ca2+ is taken up into an IP,-insensitive, presumably ryanodine-sensitive, pool. Overloading of the IP,-insensitive pool and elevation of [Ca*+], triggers release of Ca2+from this pool. In rat megakaryocytes caffeine and ryanodine, which release Ca2+from ryanodinesensitive stores, and procaine, a blocker of Ca2+ release from Ca2+-induced Ca2+ release pools, had no effect on IKcaoscillations [17], suggesting that rat megakaryocytes posses only a single IP,-sensitive Ca2+ store. Most of the remaining models can be classified as single pool models, where Ca2+is released by IP, from a single intracellular Ca2+ pool. These models appear to require some form of negative feedback, where IP, causes an initial rapid release of Ca2+ from the store, and the released Ca2+ somehow feeds back to inhibit further release of Ca2+ by IP, [62,63]. [Ca*+], oscillations in rat megakaryocytes appear to be of this type. inhibition of [Ca2+], oscillations

what are the possible mechanisms by which agents that elevate CAMP and cGMP inhibit [Ca2+], oscillations in rat megakaryocytes? Using internal dialysis of IP, from a whole cell patch pipette, it was possible to record IKea oscillations from individual rat megakaryocytes, that were inhibited by IBMX and forskolin [17]. These findings indicate that [Ca2+li oscillations in rat megakaryocytes occur in the presence of a constant concentration of IP, and that agents which elevate CAMP, and probably also cGMP, are acting down stream from the production of IP, when they inhibit these oscillations. Furthermore, these findings indicate that the falling phase of each Ca2+ spike that makes up the oscillations is not the result of hydrolysis IP,, but rather results from Ca2+ uptake or extrusion. It has been suggested that CAMP and cGMP act by inhibiting IP,-induced Ca2+release [64,65], and/or by activating Ca2+ uptake or extrusion [66]. Mechanisms of [Ca2+], wave propagation

All of the proposed mechanisms of [Ca2+li wave propagation contain some form of positive feedback but differ in whether Ca2+ or IP, is the propagating messenger [35,59,61,67]. In as much as internal dialysis of rat megakaryocytes with IP, causes IKCaoscillations [17], it seems unlikely that IP, diffusion plays a role in [Ca*+], wave propagation. Our finding that the rate of [Ca2+], wave propagation has a Qlo of 1.3 is consistent with a biological process limited by Ca2+ diffusion. Similarly a Q,, value of 1.5 was obtained for the rate of circular [Ca2+li wave propagation in Xenopus Zati oocytes [35]. We Cell Calcium

(1997)

21(5),

331-344

342

S Tertyshnikova, A Fein

found that [Caz+],waves spread with an average speed of 33.6 + 6.2 pm/s. In those non-excitable cells, where [Ca’+], waves are seen, the rate of propagation was usually in the range of 5-50 pm/s [Zl]. For cells which are morphologically and functionally polarized, [Ca2+li waves usually originate from a specific locus in the cell. For example, [Caz+],waves in hepatocytes [34] originate from a specific locus in each cell, which is the same regardless of the stimulus used. In pancreatic acinar cells, [Caz+],waves propagate from the basolateral end to the luminal region [68]. Also, in adrenal chromaffm cells stimulated with carbachol (0.3-l @I), [Caz+], begins to increase in the region beneath the plasma membrane facing the extracellular environment and propagate to the intercellular margins [69]. In other non-excitable cells, the site at which [Ca*+], waves originate depends on the site of stimulation. In mast cells, when a secretagogue is locally applied through a micropipette, the [Ca*+], wave begins at the site of stimulation from which it spread to the rest of the cell through the nucleus 1701.In hamster oocytes, the first two or three repetitive [Ca’+], transients occur as a wave originating from the site of fertilisation, while subsequent increases occur almost synchronously throughout the entire egg [71]. Injection of InsP,S, into xertm oocytes induced the release of [Caz+], from several discrete intracellular site, most of which triggered [Ca2+li waves that propagated away from the site of initiation [67]. In megakaryocytes, which do not exhibit any discernible morphological polarity, the site of [Caz+], wave initiation appears to be random. In 3 of the 20 cells investigated with fluorescence ratio imaging in this study, the point of initiation and direction of propagation of individual waves changed from spike to spike within the [Caz+li oscillation. This could be explained by assuming that the apparatus responsible for [Ca*+], wave initiation is distributed uniformly across the cell membrane. After the peak of the [Caz+], spike, [Ca*+], decayed uniformly over the whole cell. Similar spatially uniform termination of [Ca*+], waves across the cell were seen in hepatocytes [34] and aequorin-injected hamster eggs [72], which may suggest that the Ca2+ sequestration machinery is distributed uniformly across the cell. The high temperature dependence, Q,, -4, for the falling phase of the [Caz+], spike is consistent with the idea that the falling phase of each [Ca*+], spike is the result of an energy dependent process, that is Ca2+ uptake and/or extrusion by Ca-ATPases and the Na/Ca-exchanger. Accordingly, a Q10value of 3.0-5.1 was found for the Capump [73,74] and a Q,,value of 3.6-4.0 for the Na/Caexchanger [38,75]. In the preceding discussion, we suggested a role for positive feedback by Caz+to account for [Caz+],waves and negative feedback by Ca*+ to account for [Ca*+], oscillaCellCalcium

(1997)

21(5),

331-344

tions. How can it be that Ca*+ can play a role in both positive and negative feedback? The answer probably lies in the properties of the IP,-gated channel, which has been shown to have a biphasic dependence on Ca*+ at concentrations found in cells 1761. Within the physiological range of [Ca2+li, the IP,-gated channel allows positive feedback and then negative feedback for Ca*+ release. Based on the preceding discussion, we suggest that a single IP, sensitive Caz+ pool model can account for [Ca*+], oscillations and waves in rat megakaryocytes. A single pool model for [Ca*+],oscillations and waves in Xenopus oocytes was also recently proposed [77]. CONCLUSION

In response to ATP, individual rat megakaryocytes displayed concentration-dependent [Caz+], oscillations, that were reversibly inhibited by agents which elevate intracellular CAMP and cGMP. The oscillations were the result of a well-defined [Ca2+liwave, that spread across the cell with an average velocity of 33.6 * 6.2 ym/s. An analysis of the temperature dependence of [Ca*+], waves showed that rate of [Ca2+li decay during the falling phase of an individual [Caz+], spike exhibited a considerable dependence on temperature (Q,, - 4. l), whereas, the propagation velocity was relatively insensitive to temperature (Q,, - 1.3). These findings lead us to suggest that the rate limiting process for [Caz+], wave propagation in rat megakaryocytes is the rate of [Caz+],diffusion. ACKNOWLEDGEMENTS

We thank Dr Alexey Alekseev for valuable advice and the staff of the Center for Biomedical Imaging at the UCHC for helpful discussions, in particular on quantitative analysis of [Ca2+liwaves. REFERENCES

5. 6.

7.

Rink T.J.,Sage SO. Calcium signalling in human platelets. Annu Rev Pkysioll990; 52: 431-449. Sargeant P., Sage SO. Calcium signalling in platelets and other nonexcitable cells. Pkurmacol Tker 1994; 64: 395-443. Siess W. Molecular mechanisms of platelet activation. Am J Pkysioll989; 52: 58-i 78. Brass L.F.The biochemistry of platelet activation. In: Hoffman R., Benz EJJ., Shattil S.J.,Furie B., Cohen HJ. (Eds) Hematology: Basic principles and Pm&ice. New York: Churchill Livingstone, 1991; 1176-l 197 Feinman R.D., Detwiler T.C. Platelet secretion induced by divalent cation ionophores. Natnre 1975; 249: 172-l 73. Mahaut-Smith M.P., Sage S.O., Rink TJ. Receptor-activated single channels in intact human platelets. J Biol Ckem 1990; 265: 10479-10483. Sage S.O.,Rink TJ. The kinetics of changes in intracellular calcium concentration in fura- loaded human platelets. J Biol Ckem 1987; 262: 16364-16369. 0 Pearson

Professional

Ltd 7997

[Ca2’] oscillations and [C&]i waves in rat megakaryocytes

8. Sage SO., Reast R., Rink TJ. ADP evokes biphasic Caz+ infktx in fura-2-loaded human platelets: evidence for Caz+ entry regulated by the intracellular Caz+ store. Biockem J 1990; 265: 675-680. 9. Waker U. Physiological role of cGMP and cGMP-dependent protein kinase in the cardiovascular system. Rev Pkysiol Biockem Pkamzacoll989; 113: 41-88. 10. Geiger J., Nolte C., Butt E., Sage S.O., Waker U. Role of cGMP and cGMP-dependent protein kinase in nitrovasodilator inhibition of agonist-evoked calcium elevation in human platelets. Proc Natl Acad Sci USA 1992; 89: 1031-1035. 11. HaIbrugge M., Walter U. The regulation of platelet function by protein kinases. In: Huang C-K., Sha’afi RI. (Eds) Proteitt Kineses in Blood Cell Function. Boca Raton: CRC Press, 1993; 245-298. 12. Heemskerk J.W.M., Hoyland J., Mason W.T., Sage S.O. Spiking in cytosolic calcium concentration in single fibrinogen-bound fura-2-loaded human platelets. Biockem J 1992; 283: 379-383. 13. Heemskerk J.W.M., Vis P., Feijge M.A.H., Hoyland J., Mason W.T., Sage S.O. Roles of phospholipase C and calcium-ATPase in calcium responses of single, fibrinogen-bound platelets. J Biol Chem 1993; 268: 356-363. 14. Nishio H., Ikegami Y., Segawa T. Fluorescence digital image analysis of serotonin-induced calcium oscillations in single blood platelets. CeZZCalcium 1991; 12: 177-184. 15. Ariyoshi H., Sahman E.W. Spatial distribution and temporal change in cytosolic pH and calcium in resting and activated single human platelets. CeU Calcium 1995; 17: 3 17-326. 16. Uneyama C., Uneyama H., Akaike N. Cytoplasmic calcium oscillation in rat megakaryocytes evoked by a novel type of purinoceptor. J PkysioZ1993; 470: 73 1-749. 17 Uneyama H., Uneyama C., Akaike N. IntraceIIular mechanisms of cytoplasmic calcium oscillation in rat megakaryocyte. J Biol Ckem 1993; 268: 168-174. 18. Somasundaram B., Mahaut-Smith M.P. Three cation infktx currents activated by purinergic receptor stimulation in rat megakaryocytes. JPkysioZ 1994; 480: 225-231. 19. Somasundaram B., Mahaut-Smith M.P. A novel monovalent cation channel activated by inositol trisphosphate in the plasma membrane of rat megakaryoyctes. J Biol Ckem 1995; 270: 16638-16644. 20. Ikeda M., Kurokawa K., Maruyama Y. Cyclic nucleotidedependent regulation of agonist-induced calcium increases in mouse megakaryocytes. JPkysioZ 1992; 447: 71 l-728. 2 1. FewtreB C. Calcium oscillations in non-excitable cells. Annu Rev Pkysioll993; 55: 427-454. 22. Jacob R. Calcium oscillations in electrically non-excitable cells.. Biockim Biopkys Acta 1990; 1052: 427-438. 23. Ozaki Y., Yatomi Y., Wakasugi S., Shirasawa Y., Saito H., Kume S. Thrombin-induced calcium oscillation in human platelets and MEGOl, a megakaryoblastic leukemia cell line. Biockem BiopkysRes Commun 1992; 183: 864-871. 24. Kawa K. Guinea-pig megakaryocytes can respond to external ADP by activating calcium-dependent potassium conductance. JPkysioll990; 431: 207-224. 25. Grynkiewicz G., Poenie M., Tsien R.Y. A new generation of Caz+ indicators with greatly improved fluorescence properties. J Biol Ckem 1985; 260: 3440-3450. 26. Rooney T.A., Sass EJ., Thomas A.P. Characterization of cytosolic calcium oscillations induced by phenylephrine and vasopressin in single fura-2-loaded hepatocytes. J Biol Ckem 1989; 264: 17131-17141. 27 Brune B., Ullrich V. Cyclic nucleotides and intracellular-calcium homeostasis in human platelets. EurJBiockem 1992; 207: 607-613.

0 Pearson

Professional

Ltd 1997

343

28. Aktories K., Jacobs K.H. Regulation of platelet cyclic AMP formation. In: Longenecker G.L. (Ed.) The PZutelets:PkytioZugy and Pkurmacokgy. Orlando: Academic Press, 1985; 271-288. 29. Beavo J.A., Rogers N.L., Crofford O.B., Hardman J.G., Sutherland E.W., Newman E.V. Effects of xanthine derivatives on Iipolysis and on adenosine 3’,5’-monophosphate phosphodiesterase activity. MoZ PkurmacoZl970; 6: 597-603. 30. Feinstein M.B., Egan JJ., Sha’aii RI., White J. The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production (PDG,, PGE,, forskolin). Biockem Biopkys Res Commun 1983; 113: 598-604. 3 1. Siegl A.M., Daly J.W., Smith J.B. Inhibition of aggregation and stimulation of cyclic AMP generation in intact platelets by the diterpene forskolin. Mol Pkurm.acoll982; 21: 680-687 32. Geiger J., Nolte C., Waker U. Regulation of cakium mobiIization and entry in human platelets by endothelium-derived factors. Am JPkysioZ 1994; 267: C236-C244. 33. Amundson J., Clapham D. Calcium waves. Cuw opin NeurobioZ 1993; 3: 375-383. 34. Rooney T.A., Sass EJ., Thomas A.P. Agonist-induced cytosolic calcium oscillations originate from a speciiic locus in single hepatocytes. JBioZ C&m 1990; 265: 10792-10796. 35. LechIeiter J.D., Clapham D.E. Molecular mechanisms of intracellular calcium excitability in X. &ret& oocytes. CeZZ1992; 69: 283-294. 36. HiZZeB. Ionic Channels of Excituble Membranes. Sunderland: Sinauer Associates 1984; 307 37 McLarnon J.G., Wang X-P. Temperature dependence of drug blockade of a calcium-dependent potassium channel in cultured hippocampal neurons. Biopkys J 199 1; 60: 1278- 1287 38. Kimura J., Miyamae S.,Noma A. Identification of sodiumcalcium exchange current in single ventricular cells of guineapig. JPkysioZ 1987; 384: 199-222. 39. Busch A.E., Lang F. Effects of [Caz+],and temperature on minK channels expressed in Xenopus oocytes. FEBS Lett 1993; 334: 22 l-224. 40. Harootunian A.T., Kao J.P.Y.,Paranjape S., Tsien R.Y. Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP,. Science 1991; 251: 75-78. 41. Sage S.O.,Adams DJ., Van Breemen C. Synchronized oscillations in cytoplasmic free calcium concentration in confluent bradykinin-stimulated bovine pulmonary artery endothelial cell monolayers. J Biol Ckem 1989; 264: 6-9. 42. Tsunoda Y., Owyang C. Differential involvement of phospholipase A,/arachidonic acid and phospholipase C/phosphoinositol pathways during cholecystokinin receptor activated CaZ+oscillations in pancreatic acini. Biockem Biopkys Res Commun 1993; 194: 1194-1202. 43. Coombs J., Thompson S. Forskolin’s effects on transient K current in nudibranch neurons is not reproduced by CAMP. J Neurosci 1987; 7: 443-452. 44. Watanabe K., Gola M. Forskolin interaction with voltagedependent K channels in Helix is not mediated by cyclic nucleotides. Neurosci Lett 1987; 78: 2 1 l-2 16. 45. Zerr P., Feltz A. Forskolin blocks the transient K current of rat cerebellar granule neurons. Neuroscz’Leti 1994; 181: 153-l 57 46. Hoshi T., Garber S.,Aldrich R. Effect of forskolin on voltagegated K+ channels is independent of adenylate cyclase activation. Science 1988; 240: 1652-1655. 47 Berridge M.J., Irvine R.F. Inositol phosphates and cell signalling. Nature 1989; 341: 197-205. 48. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 1984; 308: 693-697 49. Ferris CD., Huganir R.L., Supattapone S., Snyder S.H. Purified

Cell Calcium

(1997)

21(5),

331344

344

S Tertyshnikova, A Fein

inositol 1,4,5-ttisphosphate receptor mediates calcium flux in reconstituted lipid vesicles. NacUre 1989; 342: 87-89. 50. Kawanishi T., Blank L.M., Harootunian A.T., Smith M.T., Tsien R.Y. CaZ+Oscillations induced by hormonal stimulation of individual fura-Z-loaded hepatocytes. J Biol C’kem 1989; 264: 12859-12866. 5 1. Gray P.T.A. Oscillations of free cytosolic calcium evoked by cholinergic and catecholaminergic agonists in rat parotid acinar cells. JPhysioll988; 406: 35-53. 52. Jacob R., Merritt J.E.,Hallam TJ., Rink T.J. Repetitive spikes in cytoplasmic calcium evoked by histamine in human endothelial cells. Nature 1988; 335: 40-45. 53. Millard PJ., Ryan T.A., Webb W.W., FewtreIl C. hnmunoglobulin E receptor cross&king induces oscillations in intracellular free ionized calcium in individual tumor mast cells. J Biol Chem 1989; 264: 19730-19739. 54. Woods N.M., Cuthbertson K.S.R., Cobbold P.H. Repetitive transient rises in cytoplasmic free calcium in hormonestimulated hepatocytes. Nature 1986; 319: 600-602. 55. Woods N.M., Cuthbertson K.S.R.,Cobbold P.H. Phorbol-esterinduced oscillations in cytoplasmic free calcium concentration in single rat hepatocytes. BiochemJ 1987; 246: 619-623. 56. Berridge MJ., Cobbold P.H., Cuthbertson K.S.R. Spatial and temporal aspects of cell signalling. Philos Tram R Sot Lond [Biol] 1988; 320: 325-343. 5% Cuthbertson K.S.R., Chay T.R. Modelling receptor-controlled intracellular calcium oscillators. Cell Calcium 199 1; 12: 97- 109. 58. Meyer T., Sttyer L. Molecular model for receptor stimulated calcium spiking. Proc Natl Acad Sci USA 1988; 85: 505 l-5055. 59. Meyer T., Stryer L. Calcium spiking. Annu Rev Biopkys Chem 1991; 20: 153-174. 60. DuPont G., Berridge MJ., Goldbeter A. Signal-induced Ca2+ oscillations: properties of a model based on Caz+-induced CaZ+ release. Cell Calcium 1991; 12: 73-85. 6 1. Goldbeter A., DuPont G., Berridge MJ. Minimal model for signal induced calcium oscillations and for their frequency encoding through protein phosphorylation. Proc NaU Acad Sci USA 1990; 87: 1461-1465. 62. Payne R., WaIz B., Levy S.,Fein A. The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback. Pkilos Trans R Sot London [Biol] 1988; 320: 359-379. 63. Payne R., Flares T.M., Fein A. Feedback inhibition by calcium limits the release of calcium by inositol trisphosphate in Limulusventral photoreceptors. Neuron 1990; 4: 547-555. 64. Tohmatsu T., Nishida A., Nagao S.,Nakashima S., Nozawa Y.

Cell Calcium

(1997)

21(5),

331-344

65.

66.

67 68. 69. 70.

71. 72.

73. 74.

75. 76.

77.

Inhibitory action of cyclic AMP on inositol 1,4,5+isphosphateinduced calcium release in saponin-permeabilized platelets. Biockim Biopkys A& 1989; 1013: 190-193. Cavallini L., Coassin M., Borean A., Alexandre A. Prostacyclin and sodium nitroprusside inhibit the activity of the platelet inositol 1,4,5-t&phosphate receptor and promote its phosphorylation. JBiol Ckem 1996; 271: 5545-5551. Kaser-Glanzmann R., Jakabova M., George J.N., Luscher E.F. Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3’,5’-cyclic monophosphate and protein kinase. Biockim Biopkys Acta 1977; 466: 429-440. DeLisle S.,Welsh MJ. Inositol trisphosphate is required for the propagation of calcium waves in Xenopus oocytes. J BioZ Chem 1992; 267: 7963-7966. Habara Y., Kanno T. Dose-dependency in spatial dynamics of [CaZ+],in pancreatic acinar cells. Cell Calcium 1991; 12: 533-542. Kanno T., Habara Y. Dose-dependency in spatial dynamics of [Ca2+lcin adrenal chromafhm cells. Cell Calcium 199 1; 12: 523-53 1. Katagiri S.,Takamatsu T., Minamikawa T., Fujita S. Secretagogue-induced calcium wave shows higher and prolonged transients of nuclear calcium concentration in mast cells. FEBS Leti 1993; 334: 343-346. Miyazaki S. Repetitive calcium transients in hamster oocytes. Cell Calcium 1991; 12: 205-216. Miyazaki S., Hashimoto N., Yoshimoto Y., Kishimoto T., Igusa Y., Hiramoto Y. Temporal and spatial dynamics of the periodic increase in intracellular free calcium at fertilization of golden hamster eggs. Dev Biol1986; 118: 259-267. Stein R.B., Gordon T., Shriver J. Temperature dependence of mammalian muscle contractions and ATPase activities. Biophys J 1982; 40: 97-102 Sunano S.,Moriyama K., Shimamura K. The relaxation and Capump inhibition in mesenteric artery of normotensive and stroke-prone spontaneously hypertensive rats. Microvasc Res 1991; 42: 117-124. Russel J.M., Blaustein M.P. Calcium efflux from barnacle muscle fibers. J Gen Physioll974; 63: 144-167 Bezprozvanny I., Watras J., Ehrlich B.E. Bell-shaped calcium response curves of Ins( 1,4,5)P,- and calcium-gated channels from endoplasmic reticulum of cerebellum. NacUre 1991; 351: 75 l-754. Atri A., Amundson J., Clapham D., Sneyd J. A single-pool model for intracellular calcium oscillations and waves in the Xenopus kxvis oocyte. Biqkys J 1993; 65: 1727-l 739.

0 Pearson

Professional

Ltd 1997