Activation of amino acid uptake at fertilization in the sea urchin egg

Experimental

Activation

of Amino

Requirement

DENIS

ALLEMAND,* Laboratoire

Cell Research

169 (1987) 169-177

Acid Uptake at Fertilization Sea Urchin Egg

for Proton Compartmentalization Cytosolic Alkalosis

GUY

During

DE RENZIS, JEAN-PIERRE PATRICK PAYAN

de Physiologie cellulaire et comparee, de Nice, 06034 Nice, Ctdex,

CNRS France

in the

GIRARD

and

UA 651, UniversitJ

The comparative importance of the release of intracellular ionic calcium, Na+/H+ exchange and cytosolic alkalosis as activator signals was studied on the development of amino acid uptake at fertilization in sea urchin eggs. We show that, once stimulated, the rate of valine uptake is greatly dependent upon intracellular pH. Suppression of the Nat/H+ exchange at the time of activation, by applying ionophore (A23187) in sodiumfree artificial sea water (ONaASW), inhibits the development of valine influx. This cannot be restored by a further (30 min later) alkalosis by transferring eggs into sea water. Suppressing the alkalosis in the presence of Na+/H+ exchange at fertilization by simultaneous addition of acid into sea water results in activation of the amino acid carrier which exhibits an increased rate of transport as soon as the eggs are replaced in sea water at pH 8.0. The absence of alkalosis in eggs activated in ONaASW can be counterbalanced either by adding NH&l 10 mM or by transfer into ASW at pH 9.0 at activation. Ammonia-treated eggs absorbed amino acid as controls, whereas eggs in sea water at pH 9.0 failed to develop a valine uptake system, suggesting that ammonia can completely replace the effect of Na+/H+ exchange. Furthermore, addition of NH&l immediately before fertilization conceals the Na+/H+ exchange but stimulates valine uptake as in controls. These data suggest that: (1) the occurrence of the intracellular calcium increase alone is not sufficient for the development of the amino acid transport system; (2) cell alkalinization at fertilization derives from the cytoplasmic membrane-located Na+/H+ exchange and an inward movement of protons into a cortical acidic compartment, which is discussed. @ 1987 Academic

Press. Inc.

Interest in an intracellular pH (pHi) shift as a possible ionic signal for cell activation was stimulated by studies on sea urchin eggs [l] and, since, it has been widely recognized that this mechanism could be extended to cell activation by growth factor or hormones [2]. During cell activation, ubiquitous alkalinization appears necessary to promote stimulation of various mechanisms which characterize an activated metabolism: oxygen consumption, enzymatic reaction, macromolecule synthesis and membrane-mediated transport process [3]. It has also become clear that mobilization of calcium from intracellular stores results from cell stimulation (see review [4]). Therefore, it seems that the Na+/H+ exchange * To whom offprint requests should be sent. Address: Laboratoire de Physiologie cellulaire comparee, CNRS UA 651, Universite de Nice, Part Valrose, 06034 Nice, Cedex, France.

et

Copyright 0 1987 by Academic Press, Inc. AI1 rights of reproduction in any form resewed 0014~4827/87 $03 .OO

170

Allemand

et al.

and the burst of ionized calcium participate together in the transduction of the external signal into a metabolic response. In sea urchin eggs we had recently shown that stimulation of the sodium pump at fertilization necessitated the occurrence of a transitory rise in cytosolic Ca*+ and of cytosolic alkalinization [5]. Nevertheless, the relative importance of each of these triggering events is not known. It is assumed that conditions which prevent the Na+/H+ exchange failed to promote the cellular processes involved at fertilization, particularly the sodium pump [5] and the amino acid uptake [6]. Furthermore, the presence in the eggs of acidic vesicles [7, 81 displaying a change in pH following fertilization, suggests that intracellular movement of protons might occur independently of the plasma membrane-located Na+/H+. Weak bases (NH&I) which directly raise pHi, while bypassing the intracellular calcium release, can mimic some of the fertilization events [9]. But recently, DubC & Epel [ 101 proposed a possible pH-unrelated mechanism by which ammonia could activate sea urchin eggs. The present study is an attempt to dissociate, first, the intracellular calcium release from the Na+/H+ exchange and, second, the latter exchange from ensuing alkalinization resulting from fertilization. Amino acid uptake, which has been extensively studied during sea urchin egg fertilization [6, 1 l-131, was chosen as an index of cell activation because, among a wide variety of membranemediated transport processes, it is strictly linked to cellular activation and growth [14, IS]. We demonstrate in the present report that the development of the amino acid transport system at fertilization requires a Na+/H+ exchange, but may be induced in the absence of resulting cytosolic alkalinization. It is suggested that, in addition to the Na+/H+ exchange, fertilization triggers an intracellular movement of protons toward a cortical compartment, which plays a key role in the activation of the membrane-located transport mechanisms. Furthermore, this phenomenon should take place simultaneously with intracellular calcium release. MATERIALS Biological

AND

METHODS

Materials

liuidus eggs were collected and maintained, as previously described 1161. Egg concentrations were adjusted to 4% per volume and kept in suspension at 20°C by stirring with a three-blade propeller. Paracentrotus

Measurement

of Amino Acid Transport

Amino-acid uptake was performed by the pulse technique for determining initial velocity [12]. Oneminute-pulse experiments were carried out with an external concentration of valine of 0.5 PM. Results are expressed as nmole of valine per hour and mg protein. The labelled precursor was [3H]valine (40 Wnmole) obtained from CEA, Saclay.

Control of External pH and Determination

of H+ Excretion

In all experiments external pH was maintained constant and acid production was determined by using an automatic titrator set-up (Tacussel Urectron 6) as described by Payan et al. [17]. Exp CellRes

169 11987)

Activation Measurement

of amino acid transport

in sea urchin egg

171

of pHi

Accumulation of DMO into eggs was used to evaluate pHi. [‘4C]DM0 (0.1 @-X/ml, final concentration 2 pM) was equilibrated into eggs for 20 min before pHi determination [ 171.

Media and Chemicals All experiments were performed by using artificial sea water (ASW) in order to avoid any contamination by exogenous amino acids [12]. Na+-free ASW (ONaASW) was made by using choline chloride as substitute for NaCl and KHC03 as substitute for NaHCO+ Measurement of internal Na+ was made, after disruption of eggs, using an Eppendorf flame photometer. Protein content was measured by the Lowry method with a Technicon autoanalyser. Ion content was expressed as uequiv/mg protein.

RESULTS

AND

DISCUSSION

In order to distinguish the relative importance of the calcium signal, Nat/H+ exchange, and cellular alkalosis for the development of a sodium-dependent amino acid transport by fertilization, we chose conditions which allowed us either to suppress or to mimic any of these events artificially. As amino acid absorption by fertilized eggs is sodium-dependent, the rate of uptake was systematically determined in sea water by rapid transfer of the eggs for the duration of the radioactive pulse. Inhibition of amino acid uptake may result from two distinct phenomena: first, a lack of development of the Naf-dependent process and, second, an inhibition of the existing transport system, the latter case being observed when the Nat gradient is reduced [ 181. It will be shown in the following section that lowering the pHi of eggs leads to inhibition of amino acid absorption. Thus, in the present paper, the modifications undergone by the sodium gradient and the intracellular pH will be taken into account before considering the significance of a possible change in amino acid uptake. Effect of Cellular Acidosis on Amino acid Absorption

Two means were used to acidify fertilized eggs 6 min after sperm contact, i.e. after complete achievement of Ca 2+ burst and Na+/H+ exchange. First, eggs were transferred into ONaASW in which they develop a cytosolic acidosis by reversing the Na+/H+ exchange [17] (fig. 1A). Second, as Johnson & Epel [193 demonstrated that the pHi of eggs is a linear function of external pH by lowering external pH from 8.0 to 7.0 6 min after fertilization, we observed a rapid decrease in pHi (fig. 1 C). In both cases, we noted considerable inhibition of valine absorption which cannot be attributed to a change in cell sodium (fig. 1 B,D). Transfer of these acidotic eggs into ASW at pH 8.0, 35 min after activation, restores normal valine uptake simultaneously with cytosolic alkalinization (fig. 1). These results indicate that the mechanism of amino acid absorption by fertilized eggs is highly pHrdependent. This pHi sensitivity has been described Exp CellRes

169 (1987)

172 4

Allemand

et al.

Valine influx

“.19)

1. Effect of cellular acidosis on valine uptake and sodium content of sea urchin eggs. Acidosis is obtained after transfer of the eggs into either ONaASW (A, B) or ASW at pH 7.0 (C, II). (A, B) Eggs suspended in sea water were fertilized at time zero (S) and divided into two batches 6 min later (arrow, I). O----O, Control, eggs maintained in ASW; m-m, eggs transferred by centrifugation into ONaASW; A-A, 35 min after fertilization, half of the eggs in ONaASW were replaced into ASW (arrow 2). (C, D) Same protocol as for (A, B) but 6 min after fertilization, eggs were transferred into sea water at pH 7.0 (arrow 1). O--O, Control; W-m, eggs in ASW pH 7.0; A-A, 35 min after fertilization, half of the eggs in ASW, pH 7.0, were replaced into ASW, pH 8.0 (arrow 2). Numbers in parentheses represent the values of intracellular PH. Fig.

min

?

for other cells concerning mainly the A system [15]. Several authors have suggested that this pH effect could be due not only to a change in pHi, but also to a change in protonation of charged sites at the outer surface of the membrane

WI. Importance

of Na+lH+

Exchange at Fertilization

In this section we examine how the lack of Na+/H+ exchange affects the stimulation of amino acid transport. Activation of eggs with A23187 in ONaASW provides a situation in which Na+/H+ exchange is obviously missing, while ionized calcium burst occurs as attested by elevation of the fertilization membrane [21, 221. Fig. 2 shows that the absence of Na+/H+ exchange during activation leads to considerable inhibition of valine absorption (fig. 2 A), while the cell Na+ decreases as in the controls (fig. 2B). However, eggs activated in ONaASW displayed a cytosolic acidosis (fig. 2A), which possesses an inhibitory effect on valine uptake. When acidosis is suppressed by transferring the eggs into sea water 30 min after activation, we did not observe any increase in the rate of amino acid uptake, although a transient rise in cell Na+ shows that a Na+/H+ Exp Cell

Res 169 (1987)

Activation Valine

of amino acid transport

in sea urchin egg

173

influx

II ,“; -p I

e

B

Fig. 2. Calcium ionophore A23187 activation of eggs suspended in ONaASW and then transferred into sea water. (A) Valine influx; (B) Na+ content. Suspension of unfertilized eggs from one female was divided equally and activated with A23187 (50 PM) either after transfer into ONaASW by centrifugation (CM), or in sea water (0-U). Thirty minutes after ionophore addition, half of the eggs in ONaASW were replaced into sea water (A-A). Numbers in parentheses represent intracellular pH.

4. 6 5.\\ a ‘\

0.2

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0

30

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60

min

exchange does occur at the time of the transfer [5]. These results indicate that (1) the Na+/H+ exchange and/or the resulting alkalosis are obligatory events that ensure development of the amino acid transporter; (2) this exchange must occur simultaneously with the early events that accompany egg activation: exocytosis and, more likely, the cellular calcium increase. Effect of Suppressing Cytosolic Alkalosis at Fertilization

This section presents the experimental conditions under which egg alkalinization occurring at fertilization is suppressed by simultaneous acidification of the external medium within sperm addition. Fig. 3 reveals that a decrease of the

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Valine

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3. Effect of simultaneous acidification of external medium (A) during fertilization, on cellular pH (B), Na+ content (C) and vahne infhrx (0). A-A, 40 ml of 4% egg suspension was fertilized (S) in sea water and, immediately after sperm addition, 250 pl of HCI, 10 mM was progressively added into the external medium. A-A, 20 min post-fertilization, half of the eggs were replaced into ASW at pH 8.0 (2).

Fig.

influx

30

* 60 min

Exp Cell

Res

169 (19873

174

Allemand Valinc

OJ

,

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et al.

influx

30

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do

min

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content

Fig. 4. Effect of weak base addition during calcium ionophore activation of eggs suspended in ONaASW. (A) Valine uptake; (B) cell Na+. m-m, Eggs were activated in ONaASW with 50 pM A23187 plus 10 nM NH&l (arrow); U-0, control, activation of eggs with A23187 (50 PM) in sea water (arrow). Intracellular pH is indicated in parentheses. 0-0

min

external pH from 8.0 to 7.0 by a progressive addition of HCl to sea water, after the sperm addition and during a time corresponding to the development of the Na+/H+ exchange, allows one to maintain the intracellular pH of the egg at the unfertilized level (fig. 3 A, B). The transient increase shown by cell Na+ indicates that the Na+/H+ exchange had occurred normally, although it was not possible to titrate the appearance of corresponding external protons (fig. 3 C). As long as pHi is artificially maintained at the unfertilized level, the activity of the amino acid carrier is blocked (fig. 30). However, this inhibition may be reversed by increasing the external pH to 8.0 (fig. 3). This demonstrates that the transport mechanism for amino acid was triggered by sperm, but became effective only when alkalinization was achieved. Therefore, a contradictory point is raised by these results: it appears that the Na+/H+ exchange is a necessary event for stimulation of amino acid absorption, but that the usually associated cytosolic alkalosis may be prevented without an inhibitory effect on the setting-up of the amino acid transporter. Thus, we can hypothesize that, beside the cytoplasmic alkalinization, the Na+/H+ exchange is responsible for another triggering mechanism which must necessarily occur at the time of the early events of fertilization. Separated Effects of Na+lHf

Exchange and Intracellular

Alkalosis

In the experiments described below we provided conditions in which fertilization-induced alkalosis is mimicked, although Nat/H’ exchange does not occur. Eggs were transferred in ONaASW and activated with calcium ionophore A23187, alkalosis being obtained by simultaneous addition of 10 mM NH&l (fig. 4) or by an elevated external pH (fig. 5). Both cases led to a cell pH rise comparable to that we observed after fertilization. Comparison of figs 4 and 5 shows that addition of NH&l allows egg activation to develop a high rate of valine absorption (fig. 4), while eggs activated in alkaline media failed to pump Exp Cell

Res 169 (1987)

Activation uallnr

of amino acid transport

in sea urchin egg

175

influx

4m) A

.

/

0.3

1

6c ? a <0

I .

Fig. 5. Calcium ionophore activation of eggs in ONaASW; effect of alkalinized sea water (pH 9.0). (A) Valine uptake; (B) cell Na+. O-0, Eggs maintained in ONaASW were activated with 50 uM A23187 and an appropriate amount of KOH was added simultaneously to raise the external medium up to pH 9.0; m-m, control as in fig. 4. Numbers in parentheses represent intracellular pH.

0.2

.g z iL

!

0.1

valine (fig. 5). In both cases intracellular pH and the Nat gradient behave as they do after fertilization in sea water (see control). These results confirm the existence of a pH-unrelated mechanism triggered by ammonia, as recently suggested by DubC & Epel [lo], and which allows a weak base to completely mimic the effect of Na+/H+ exchange. However, the nature of such a mechanism remains unidentified. In the next experiment, 10 mM NH&l was added to eggs suspended in sea water and fertilization was performed immediately afterwards. We observe that the uptake of valine rises as in control (fig. 6A) but that Na+ exchange does not occur, as is indicated by the time course of cell Na+ which does not display a transitory peak after sperm addition (fig. 6B).) The replacing of the sperm effect (Na+/H+) by ammonia could be explained in view of the results of Winkler &

4,

Vmline

influx

No+content

OJ 0’

12-878333

20

40 min

Fig. 6. Effect of previous activation of eggs by ammonia before fertilization on valine uptake (A) and Na+ content (B). A-A, 10 mM NH&l was added to the egg suspension 10 set before fertilization (arrour). A-A, Control, eggs fertilized in sea water.

0.1 Olbio min

Exp

Cd

Res

169 (1987)

176

Allemand

et al.

SW A

C Na+

0

TO H+

H+

Ii* a”

em

sperm-

PHif

b

0

H+

Fig. 7. Diagrammatic representation of the mechanisms participating in egg alkalinization during fertilization. (A) Unfertilized eggs; (II) fertilization; (C) fertilized eggs. The size of the H’ symbol in acidic vesicles schematically represents the amount of protons sequestered in the vesicles. SW, sea water; em, egg membrane; LZU,acidic vesicle.

Grainger [9] which showed that its unprotonated form enters the eggs faster than the overall cell pH increase. We therefore propose that a cortical-located alkalosis occurs initially and leads to an inhibition of the Na+/H+ transporter, as is demonstrated in other cell types [22]. These results suggest that, as for ammonia, sperm acts not only through cell alkalosis promoted by Na+/H’, but also by means of another associated triggering effect. Proton Compartmentalization

at Fertilization

By using a fluorescent probe, Lee & Epel [7] and Christen [8] have visualized the presence of acidic vesicles in the cortical areas of unfertilized eggs. Although both of these authors envisaged that fertilization triggers modification of intravesicular pH, their conclusions remained contradictory about the evolution of this parameter. Recent experiments (unpublished results) carried out in our laboratory with Paracentrotus liuidus eggs consisted of measuring the accumulation of weak base by an isolated cortex preparation [24], [14C]methylamine being used at tracer dose. We have shown that cortices prepared 8 min after fertilization accumulate the tracer to a greater extent (2-3-fold per cortex surface) than unfertilized preparation, thus indicating that fertilization acidifies a cortical compartment, as previously suggested by Lee & Epel[7]. We may conclude from these results that cell alkalinization triggered by fertilization originates from two phenomena: a cytoplasmic membrane-located Naf/Ht exchange and inward movement of H+ into an acidic compartment; such a hypothesis is presented in the diagram in fig. 7. Furthermore, the fact that cytosolic alkalosis promoted by an increase in external pH fails to activate egg metabolism (see the present paper and [lo]) raises two points. First, the two mechanisms presented above (Naf/Hf exchange and the Ht entry into vesicles, fig. 7) appear to be dependent of each other and, second, cellular alkalosis cannot represent the link between them, which remains to be identified. Considering now all the processes triggered by ammonia in eggs, one can envisage that beside cytosolic alkalinization, weak base sets up a more favorable gradient for proton concentration in cortical vesicles; the latter effect might Exp

Ceil

Res 169 (1987)

Activation

of amino acid transport

in sea urchin egg

177

be one of the additional mechanisms suggested by DubC & Epel [lo], but it could hardly be considered as pH-unrelated. Nevertheless, when comparing the kinetics of ammonia entry into eggs [9] and that of cytoplasmic pH rise [9], which is far slower, it may be surmised that the weak base-induced entry of protons into cortical vesicles occurs immediately before cell alkalinization is achieved. Hence, we can hypothesize that, after fertilization, the cortical vesicles are acidified by the occurrence of a similar intake of protons which leads to marked alkalinization around the newly acidic vesicles, i.e., near the cortical zone. This intracellular movement of protons cannot be detected by titration at fertilization and its simultaneity with other sperm-provoked events would contribute to their participation in the efficient development of membrane transport systems, such as the sodium pump [5] or amino acid uptake. Furthermore, numerous parameters are favorably reinforced by cortical-located alkalinization: increase in membrane fluidity [25], polymerization and actin filament bundle formation [26, 271. This research was partially funded by the CNRS (UA 651) and by the Ministry of Education. We wish to thank the CEA for facilitating the purchase of radioactive products and Drs B. Lahlou and C. Ungar for their comments and suggestions on the manuscript.

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in Sweden

Exp

Cell Res

169 (1987)