Regulation of Calcium Influx in Chara

Received for publication December 18, 1991 Accepted April 25, 1992

Plant Physiol. (1992) 100, 637-643 0032-0889/92/1 00/0637/07/$01 .00/0

Regulation of Calcium Influx in Chara' Effects of K+, pH, Metabolic Inhibition, and Calcium Channel Blockers Robert J. Reid* and F. Andrew Smith Department of Botany, University of Adelaide, Box 498, G.P.O. Adelaide, S.A. 5001, Australia MATERIALS AND METHODS

ABSTRACT Measurements were made of 45Ca influx into isolated internodal cells of Chara corallina and also into internodal cells of intact plants. 45Ca influx was closely related to growth. In rapidly expanding internodal cells, the influx was approximately 1.4 nmol m-2 s-' compared to the influx in mature cells from slow-growing cultures of 0.2 nmol m-2 s-1. Isolated internodal cells had influxes in the range 0.2 to 0.7 nmol m-2 s-i, but this increased to approximately 2 nmol m-2 s-1 in high calcium solutions and to 4 nmol m-2 s-' in high potassium solutions. No significant effects on calcium influx were observed for changes in external pH or for treatments that changed internal pH, except that NH4 was slightly inhibitory. Severe metabolic inhibition by carbonylcyanide-m-chlorophenylhydrazone stimulated influx, whereas dicyclohexylcarbodiimide had no effect and darkness inhibited influx. La3" also inhibited influx, but the organic channel blockers nifedipine and bepridil stimulated influx. Verapamil had no effect. The results are generally consistent with voltage regulation of calcium channels as in animal cells.

Plant Material The giant alga Chara corallina was grown in the laboratory in large plastic tanks on a substrate of garden soil and river sand that was supplemented with an APW2 containing 1 mm NaCl, 0.1 mm K2SO4 and 0.5 mm CaSO4. The cultures were illuminated on a 16 h/8 h light/dark cycle at an intensity of approximately 50 ,umol m-2 s-' at the surface of the solution. Before experiments, individual internodal cells (40-90 mm long and approximately 1 mm in diameter) were isolated from the plant and stored overnight in APW buffered at the experimental pH with 5 mm Mes (pH 5), Mops (pH 7), Nhydroxyethylpiperazine propane sulfonic acid (pH 8), ncyclohexoaminoethane sulfonic acid (pH 9.2) or 3-cyclohexylaminopropane sulfonic acid (pH 10.4) and adjusted to the required pH with NaOH. When intact plants were used, they were taken from the culture tank and pretreated in APW at pH 7 for only 1 h before use. Unless stated otherwise, experiments were done at pH 7.

Flux Measurements We used three methods to estimate 45Ca fluxes into the cell, cytoplasm and vacuole, and in each method, it was necessary to separate the cell wall from the cell contents in a manner that allowed minimal contamination of extracellular 45Ca with intracellular calcium pools. A description of the methods used to measure fluxes of 45Ca in Chara and an outline of the general problems associated with accurately determining calcium influx into turgid plant cells is given in ref. 22. The usual tracer method of loading whole cells and then rinsing off most of the extracellular radioactivity was used for long influx times with long rinse times to estimate flux to the vacuole and slowly exchanging cytoplasmic compartments. The rinse solution was APW + 2 mm LaCl3. The purpose of the La3+ was to displace 45Ca from the cell wall and to block calcium channels to prevent any further accumulation of 45Ca. Following the rinse period, the cell was blotted and allowed to wilt slightly. The ends of the cell were removed and a hypodermic syringe was inserted into one end and clamped in position with forceps. The vacuole was displaced by injecting an air bubble through the cell and the cytoplasm was then flushed out by rapid injection of 1 mL

Fluctuations in the level of free calcium in the cytoplasm have been implicated in the control of a range of intracellular and membrane-related processes in plants (see reviews by Hepler and Wayne [12] and Kauss [14]). The relative importance of membrane transport compared to cytoplasmic buffering of calcium in determining the level of free calcium under a given set of conditions is at present impossible to assess, partly because so little is known about the magnitude of calcium fluxes in plants, and in particular about the control of calcium fluxes at the plasma membrane. This has been largely due to the considerable difficulties, common with all divalent cations, in distinguishing between extracellular binding in the cell wall and actual uptake to the cell. Recently, we reported the results of a detailed investigation of Ca2" exchange in the cell wall of Chara corallina (22), from which methods were developed for the measurement of both longand short-term fluxes of Ca2". These methods have now been refined, and in this article, we present the first survey of the effects of a range of treatments on 45Ca influx into a plant cell in an attempt to reveal the factors that control the permeability of calcium channels in the plasma membrane.

'Abbreviation: APW, artificial pond water.

'This work was supported by the Australian Research Council. 637

REID AND SMITH

638

of distilled water through the lumen, leaving a clear sleeve of cell wall. The 45Ca activity in the vacuolar and cytoplasmic fractions was determined by liquid scintillation counting. For shorter influxes, the technique that will be referred to as 'segment loading' was used. A single Chara internodal cell was mounted in a three-chambered perspex block in which the ends of the cell were isolated from the central portion by means of grease barriers. 45Ca in APW was added to the cell segment in the center for the influx period of 30 min, after which it was rinsed away by 6 to 10 changes of APW + 2 mM LaCl3 over 3 to 5 min. The cell was then removed from the chamber and fractionated as described above. These two methods measure different fluxes, and it is important to understand what is actually being measured in each case. 'Whole cell loading' is used to measure 45Ca influx from the extemal solution to the vacuole and slowly exchanging cytoplasmic compartments. For long influx times, the specific activity of 45Ca in the cytoplasm will be similar to that in the external solution, so that the vacuolar 45Ca activity will reflect the Ca2" flux from the cytoplasm to the vacuole. Segment loading, i.e. short influx and short rinse times, is used to obtain the unidirectional influx at the plasmalemma. At high external pH (>8), problems were encountered with both segment loading and whole cell loading because of the precipitation of insoluble La` salts during the rinsing period. To overcome this problem, an alternative method referred to as 'half cell loading' was employed. For this method, an internodal cell was mounted in a two-chambered perspex block with the two halves isolated by a grease barrier. 45Ca was applied to one half only for 2 to 3 h, after which the solution in both ends was sucked out and the cell removed from the chamber and blotted. The half cell from the nonradioactive end was excised and flushed as for segment loading. The validity of this technique relies on efficient distribution of 45Ca from the uptake end to the whole cell, and it is only useful over relatively long uptake periods and for cells with normal protoplasmic streaming (22). The fluxes are expressed as mean ± SE of 7 to 10 cells. The significance of the difference between means was tested using the Wilcoxon Rank Sum test as described in Sokal and Rohlf (25) for a probability of P < 0.1. RESULTS Measurement of Influx

Figure 1 shows a time course of uptake of 45Ca from the external solution to the cytoplasm and to the vacuole measured by the segment loading method with a 4-min rinse in APW containing La3+. For influx times less than 30 min, 45Ca labeling of the cell was dominated by 45Ca in the cytoplasm. Tracer influx to the cytoplasm showed two distinct phases: an initial influx lasting approximately 15 min, followed by a slower influx that was linear up to at least 100 min. Influx to the vacuole showed a lag over the first 5 to 15 min, but after 100 min, the 45Ca activity in the vacuole and cytoplasm were similar. The variability of influx (as a percent of the mean) was much higher at short influx times. After 5 min, the 45Ca activity in the cell was small in comparison to the residual 45Ca activity in the cell wall after the short rinse, so that even

Plant Physiol. Vol. 100, 1992

1.2 N

.9

1.0

-l

0.8 co 4n

0.6 0.4

Le)

0.2 M'.I

0

0

20

40 80 60 Influx time (min)

100

Figure 1. Time course of 45Ca influx into internodal cells of Chara measured using the "segment loading" method. Cells were rinsed for 4 min in APW + 2 mm LaCI3; n = 14 cells/point.

a small degree of contamination during cell fractionation would significantly increase the measured flux. The vacuolar flux was more accurately determined by loading whole cells for much longer periods, so that the vacuolar activity would be high enough not to be significantly affected by contamination during sampling. The slow exchange at the tonoplast and the large pool size of calcium in the vacuole (22) mean that long rinse periods will not result in significant loss of vacuolar 45Ca. Figure 2 shows an experiment in which cells were incubated in 45Ca for 3 h and then rinsed in La3` solution. The 45Ca activity in the vacuole was constant for rinse times greater than about 0.5 h and independent of the "5Ca in the cell wall, which continued to fall over several hours. After 4 h, rinsing the 45Ca in the cell wall was exchanging only slowly and the total activity in the wall was similar to that in the combined intracellular fractions. It is also worth noting that for long rinse times, a significant proportion of the cellular 45Ca remained in the cytoplasm

(Fig. 2). Calcium Influx and Growth The relationship between influx and growth was examined by incubating whole plants in 45Ca-APW and then rinsing and fractionating the cells as for individual isolated internodal cells. Figure 3 shows the 45Ca influx to the vacuole for internodal cells of plants from two different cultures, one of which was growing only slowly and the other was in a phase of rapid growth. C. corallina normally grows by expansion of the uppermost internodes; increase in length is greatest in the first internode from the terminal shoot, less in the second internode, and by the third internode, cells are virtually mature. It can be seen from Figure 3 that there is a strong tendency for expanding cells to have higher calcium influxes than mature internodes and for the faster growing plants to have higher influxes than slower growing plants. Because of the large variability in influx and the small size

CALCIUM INFLUX IN CHARA

639

-

0.50

0.70

0.22

0.40

1.46

-

Cell

0.30 r

0.80 0 a)

0.20 Vacuole 0.10

0.96 0

0

1

2

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0.48

0.64

4

2.0

0.38-

.-1

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eq

0

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0

I*

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Figure 3. 45Ca influx to the vacuole of internodal cells of intact plants of C. corallina from two different cultures. The numbers refer to the influx of the adjacent internodal cell in nmol m-2 s-'. Plants were incubated in 45Ca-APW for 5 h and then rinsed for 1 h in La3+APW.

Wall

0.4

0

Fast growing culture

Slow rowing cuture

L ,.1

0

1

-1

2

3

4

Rinse time (h) Figure 2. The measured 45Ca influx to the vacuole, cytoplasm, and cell wall as a function of the rinse time in La3`-APW. Whole internodal cells were incubated in 45Ca-APW for 3 h.

of the first two internodes, experiments with isolated internodes were normally conducted with larger mature cells from internodes three to five from the top of the plant. For these cells, the mean influxes measured by segment loading over 30 min lay in the range from 0.20 to 0.72 nmol m-2 s-. In hindsight, the differences appear to be related to the rate of growth of the culture, but there was also a tendency, more marked in cells with higher influxes, for the influx to decline with time after cutting.

2

1.6 -

0

1.2

N

0.8

Effects of K' and Ca21 on Calcium Influx Figure 4 shows the response of 45Ca influx to increasing Ca2+ concentration in the bathing medium. Influx doubled between 0.2 and 1 mm, was insensitive to Ca2' between 1 and 4 mm, and then increased further by a factor of 3 between 4 and 20 mM. 45Ca influx was insensitive to the extemal K+ concentration between 0.1 and 5 mM (Fig. 5, data not shown for 0.1 mM), but was greatly stimulated at higher concentrations. The maximum influx obtained at 20 mm was nearly 8-fold higher than at 1 mm. Influx decreased between 20 and 100 mm K+.

a C.

N1

0.4

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l

l 0.3

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[Ca.] (mM) Figure 4. Dependence of 45Ca influx on the calcium concentration in the bathing solution. Influx measured by segment loading. Influx time = 30 min; rinse time = 4 min.

Effects of pH and ATP Concentration

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REID AND SMITH

640

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The effects on 45Ca influx of treatments that alter intracellular pH or the pH gradient across the plasma membrane are shown in Table I. There was no effect of changing external pH or of application of butyric acid at concentrations that would be expected to acidify the cytoplasm by up to 1 pH unit (23). There was a significant inhibition of 45Ca influx by NH4, but not by methylamine. Because NH4 and methylamine normally have similar effects on cytoplasmic pH, it seems unlikely that the reduction in influx in the presence of NH4 is solely a response to changing intracellular pH. Metabolic inhibition by carbonylcyanide-m-chlorophenylhydrazone resulted in a 214% increase in influx (Table I). This was accompanied by a slowing of the streaming rate from 76 ± 2 to 6 ± 6 jim s-', which is consistent with a fall in cellular ATP content of approximately 90% (24). However, dicyclohexylcarbodiimide, which reduced the streaming rate to 34 ± 4 um s- had no significant effect on influx. It should be noted that metabolic inhibitors could increase net Ca2+ uptake without inhibiting influx if the reduced ATP concentration slowed the active efflux of Ca2' at the plasma membrane. Darkness, which reduces the ATP concentration in Chara by 10 to 20% (24) and slightly acidifies the cytoplasm (21), inhibited 45Ca influx by nearly 50% (Table I). ,

0 1

1

10

100

IK+1 (mM) Figure 5. Effect of K+ concentration on 45Ca influx to internodal cells of C. corallina measured by segment loading. Cells were pretreated in solutions of different K+ concentrations for 1 h prior to influx. Influx time = 30 min; rinse time = 4 min.

Channel Blockers Although the uptake of calcium by mature isolated internodal cells was generally quite low by comparison with actively expanding cells, there was still a considerable reduc-

Table I. Effects of Various Treatments on Calcium Influx into Internodal Cells of Chara corallina 45Ca influx Pretreatment Percent Conditions time min

Metabolic inhibitors CCCP' (0.01 mM), pHs DCCDb (0.05 mM) Amines Methylamine (0.2 mM) NH4 (0.2 mM) Weak acid Butyrate (0.5 mM), pHs (2 mM), pHs Light/dark Light -- dark External pH pH 5 pH 7 pH 9.2 pH 10.4

Control

Treatment

nmol m-2 s-

30 120

0.44 ± 0.10

0.94 ± 0.16 0.52 ± 0.16

214 nsc

240 240

0.54 ± 0.16

0.44 ± 0.10 0.20 ± 0.04

ns 37

30 30

0.46 ± 0.08

0.38 ± 0.06 0.36 ± 0.10

ns ns

60

0.44 ± 0.06

0.24 ± 0.04

55

0.22 ± 0.04d 0.20 ± 0.04d 0.22 ± 0.04d 0.28 ± 0.06d a b DCCD, Dicyclohexylcarbodiimide. CCCp, Carbonylcyanide-m-chlorophenylhydrazone. d c ns, Not significant. These experiments were done using the half cell loading technique, which does not depend on La3+ rinsing, because the latter is not possible at pH > 8 because of the insolubility of La3+ salts. All other experiments were done by segment loading. 60 60 60 60

641


tion

in

influx following addition of low concentrations of

La3" (Table II). Higher concentrations of La3" did not reduce the flux further, but acted more quickly. The La3"-insensitive flux measured by segment loading was around 0.14 nmol m-2 s-i compared to the lowest influxes in untreated cells of 0.20 nmol m-2 s-1 and more than 4 nmol m-2 s-1 in cells in high K+ solution (Fig. 5). The organic channel blockers nifedipine, bepridil, and verapamil did not inhibit 45Ca influx (Table II). Verapamil had no effect, whereas bepridil and nifedipine actually increased the influx. Verapamil and bepridil caused cells to die if applied at 0.05 mm for more than 30 min.

DISCUSSION Flux Methods

The methods normally used to measure tracer fluxes in turgid plant cells are not feasible for ions whose binding in the cell wall represents a significant proportion of the cellassociated radioactivity. For Chara, 45Ca in the cell wall exceeds that in the cell for 4 h of rinsing, and it is likely that during this time, fast exchanging cytoplasmic compartments are lost from the cell, leading to an underestimate of the plasma membrane flux. It is possible with charophytes to separate the cell wall from the contents, but at short rinse times, there appears to be significant contamination of the cytoplasm and vacuole from extracellular 45Ca. We have shown previously (22) that most of this contamination occurs via contact of cell sap and cell wall at the cut ends during insertion of the syringe needle, which was used to blow out the vacuole and flush out the cytoplasm. However, with segment loading, a syringe can be introduced into an uncontaminated end portion of the cell and the cell contents removed quickly to minimize exchange with wall-bound calcium. We found this method to be unreliable for rinse times of 1 min or less (22), but after 4 min of rinsing, contamination appears to be reduced to an acceptable level.

Ca2' Fluxes Across the Plasma Membrane and Tonoplast There has been some doubt expressed about the feasibility of measuring unidirectional influxes of Ca2' across the plasma membrane (see, for example, Wrona et al. [28]) because the small pool size of free Ca2+ in the cytoplasm might

equilibrate too rapidly with 45Ca in the external solution. Efflux of 45Ca would then cause the influx to be underestimated. The plasma membrane influx will only be significantly underestimated if (a) the cytoplasmic free Ca2" becomes labeled rapidly and (b) the efflux from the cell is large compared to fluxes to intracellular compartments. Although there may be only a very small pool of free Ca2" in the cytoplasm (approximately 0.2 yM [17]), it is likely that this pool is buffered by a much larger amount of Ca2" bound in the free cytoplasm, possibly as much as 60 /.M (1). In addition, there appears to be a larger amount of Ca2" in a more slowly exchanging pool, possibly within cytoplasmic organelles. 4"Ca accumulation in the cytoplasm (Fig. 1) indicated a moderately fast phase with a content of less than 20 gM, and a slower phase with at least another 40 glM (based on a surface area/ volume ratio of 5 x 103 m2/m3 and assuming 5% cytoplasm). The specific activity of a small pool of Ca2" in the cytoplasm that is exchanging with large pools of nonradioactive Ca2" in the ground cytoplasm, organelles, and vacuole, as well as with radioactive Ca2+ in the external solution, will be determined by the ratios of the radioactive and nonradioactive fluxes/exchanges. Our data do not indicate rapid equilibration of cytoplasmic Ca2+ with 45Ca in the external solution. Moreover, the lag in the development of the vacuolar influx is inconsistent with rapid equilibration. In relation to the size of the efflux from the cell, we reported previously that the efflux at the plasma membrane was of a magnitude similar to the influx from the cytoplasm to the vacuole. There is no evidence from the current work to support a large efflux from the cell or rapid cycling of Ca2" across the plasma membrane. Efflux must necessarily proceed against a considerable electrochemical gradient across the plasma membrane. The gradient for transport across the tonoplast is considerably smaller because of the smaller electrical potential difference across this membrane. Having considered these arguments, we are reasonably confident that the early part of the time course shown in Figure 1 reflects the unidirectional influx across the plasmalemma. The influx measured in the first 15 min of 0.36 nmol m-2 s-' is slightly higher than was reported previously using the half cell loading method (22). After 30 min, the influx was lower by 33% than that measured at 5 to 15 min. Some of the reduction may be due to the decreasing effect of residual cell wall contamination as the intracellular 45Ca

Table II. Effects of Channel Blockers on Calcium Influx into Internodal Cells of Chara corallina All fluxes measured over 30 min using segment loading with a 4- to 5-min rinse. Verapamil and LaCI3 experiments were conducted in low light (5 Amol m-2 s-'). The light intensity in the bepridil experiment was approximately 45,umol m-2 s-'. ns, Not significant. Conditions

Time min

Verapamil (0.05 mM)

45Ca Influx

Pretreatment Control

Treatment

Percent

nmol m-2 S-1

LaCI3 (0.10 mM)

20 60

0.24 ± 0.04 0.48 ± 0.14

0.28 ± 0.10 0.14 ± 0.04

ns 29

Nifedipine (0.05 mM) Low light Darkness Bepridil (0.05 mM)

20 0 0

0.36 ± 0.02 0.24 ± 0.04 0.24 ± 0.03

0.42 ± 0.12 0.62 ± 0.12 0.88 ± 0.40

ns 258 200

642

REID AND SMITH

activity increased, whereas some efflux of label is also possible. The flux of 45Ca from the external solution to the vacuole was approximately 0.1 to 0.2 nmol m-2 s-1 (Figs. 1 and 2). The actual Ca2" flux across the tonoplast will be higher than these values by the ratio of the specific activity of 45Ca in the external solution to the specific activity of 45Ca in the free cytoplasm; i.e. if the specific activity of Ca21 in the cytoplasm was half that in the external solution, the tonoplast influx would be double the tracer influx, or 0.2 to 0.4 nmol m-2 s-1, which is similar to the plasma membrane influx.

Calcium Influx and Growth Calcium influx to the vacuole in Chara appears to be closely related to growth. Expanding cells have high influxes, but in rapidly growing plants, mature internodal cells further down the plant also have higher influxes than in slow-growing plants. This implies that symplasmic transport of calcium to the shoot may also be important. In Chara, there is relatively free access between adjoining cells for small solutes (3, 20, 29), and long distance transport is aided by protoplasmic streaming. Ding et al. (5) have recently demonstrated polar transport of photoassimilates in Chara. However, plasmodesmata close almost immediately after internodal cells are isolated (20), and it is, therefore, not surprising that calcium influx decreases with time after cutting.

Regulation of Calcium Influx There is a large electrochemical gradient for calcium entry to the cell, and calcium influx as a passive uniport through membrane channels, as in animal cells, seems the most likely mechanism. The question of whether there are specific channels for Ca2', and if so how much of the flux is through these channels, has not been resolved by the current study. If the flux is predominantly mediated by channels, then it is apparent that most of the channels that pass Ca2+ in C. corallina are closed under normal conditions. We have identified high external concentrations of Ca2' and K+ as conditions that increase the permeability of the plasma membrane to calcium. The correspondence between the K+ concentration required to stimulate calcium influx and the concentration of K+ that would normally depolarize the plasma membrane (refs. 2, 15; R. J. Reid, unpublished results) point to voltage control of calcium influx, as in animal cells (17). However, there are some inconsistencies in the hypothesis that voltage is the dominant factor regulating calcium influx through channels in Chara. The small effects on calcium influx of carbonylcyanide-m-chlorophenylhydrazone, the lack of response to dicyclohexylcarbodiimide, and the fall-off in influx at very high K+ concentrations are all difficult to reconcile with voltage control of calcium channels. A detailed investigation of membrane potential difference and conductance under conditions that favor calcium influx is clearly indicated. Of the other possible control factors, severe metabolic inhibition increased the influx, whereas darkness decreased the influx. However, these changes were modest in comparison to those caused by K+ and Ca2" . In the light of a body of literature on plant cells in which changes in cytoplasmic


pH were correlated with changes in intracellular free calcium (7-11, 18), with a general tendency for pCa to increase as pHcy, increased and vice versa, the effect on 45Ca influx of treatments known to substantially alter cytoplasmic pH in Chara was examined. Application of weak acids and bases or altering external pH (Table I) had very little influence on influx, and it must therefore be concluded that changes in free calcium in the cytoplasm with changes in pHcyt are intracellular in origin, quite possibly the effect of competitive binding of H+ to calcium buffers in the cytoplasm. The higher calcium2" influxes into expanding cells indicate either that there is a higher density of channels or that channels are open more often, or a combination of the two, than in mature cells. Channel Blockers The organic channel blockers nifedipine, verapamil, and bepridil have been shown to be effective in blocking calcium channels in animal cells (13, 27), and there have been a number of reports of effects of nifedipine and verapamil on various aspects of the physiology of plant cells (e.g. refs. 3, 4, 6, 19). MacRobbie and Banfield (16) obtained variable effects of nifedipine on 45Ca influx in Chara, but we have previously shown (22) that their flux methods do not distinguish between fluxes into cells and extracellular binding, and their calcium fluxes were an order of magnitude higher than those reported here, suggesting considerable extracellular contamination. Tester and MacRobbie (26) noted inhibition by nifedipine of the inward current during the action potential in Chara, which was consistent with blockage of calcium channels in the plasma membrane. They found no effect of methoxyverapamil and a stimulation of the inward current by bepridil. We found that none of these agents reduced calcium influx. Verapamil and bepridil were toxic to cells, but this is unlikely to be due to reduced calcium influx because cells remain viable for at least several weeks in solutions containing La3" at concentrations high enough to substantially reduce calcium influx. Bepridil and verapamil clearly have severe side effects in Chara that are unrelated to their perceived mode of action, and caution should be exercised with other plant cells in interpreting responses to organic channel blockers in terms of inhibition of calcium influx; on the basis of the results shown above, a stimulation of influx is more likely. ACKNOWLEDGMENTS

This work was supported by the Australian Research Council. The authors wish to thank Patrick Kee for his excellent technical assistance and M. Tester for useful discussions on the experiments and comments on the manuscript. LITERATURE CITED 1. Baker PF, Dipolo R (1984) Axonal calcium and magnesium homeostasis. Curr Top Membr Transp 22: 195-247 2. Beilby MJ, Blatt MR (1986) Simultaneous measurements of cytoplasmic K+ concentration and the plasma membrane electrical parameters in single membrane samples of Chara corallina. Plant Physiol 82: 417-422 3. Bostrom TE, Walker NA (1975) Intercellular transport in plants.


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