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NATHALIE GRANDIN and MICHEL CHARBONNEAU. Laboratoire de Biologie et ...... BUSA, W. B., FERGUSON, J. E., JOSEPH, S. K., WILLIAMSON, J. R..
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Development 112, 461-470 (1991) Printed in Great Britain © The Company of Biologists Limited 1991

Intracellular pH and intracellular free calcium responses to protein kinase C activators and inhibitors in Xenopus eggs

NATHALIE GRANDIN and MICHEL CHARBONNEAU Laboratoire de Biologie et Ginitique du Diveloppement, URA CNRS if 256, University de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France

Summary Cell activation during fertilization of the egg of Xenopus laevis is accompanied by various metabolic changes, including a permanent increase in intracellular pH (pHi) and a transient increase in intracellular free calcium activity ([Ca 2+ ]|). Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, J. Cell Biol. 108, 885-892, 1989). Indeed, activators of PKC trigger cortical granule exocytosis and cortical contraction, two events of egg activation, without, however, releasing the cell cycle arrest (blocked in second metaphase of meiosis). In the egg of Xenopus, exocytosis as well as cell cycle reinitiation are supposed to be triggered by the intracellular Ca 2+ transient. We report here that PKC activators do not induce the intracellular Ca 2+ transient, or the activation-associated

increase in pHi. These results suggest that the ionic responses to egg activation in Xenopus do not appear to depend on the activation of PKC. In addition, in eggs already pretreated with phorbol esters, those artificial activators that act by releasing Ca 2+ intracellularly, triggered a diminished increase in pHi. Finally, sphingosine and staurosporine, two potent inhibitors of PKC, were found to trigger egg activation, suggesting that a decrease in PKC activity might be an essential event in the release of the metaphase block, in agreement with recent findings on the release of the prophase block in Xenopus oocytes (Varnold and Smith, Development 109, 597-604, 1990).

Introduction

accompanied by an increase in pHi (Webb and Nuccitelli, 1981). However, unlike that in cultured mammalian cells and sea urchin eggs, the activationassociated increase in pHi in Xenopus eggs is not due to a Na + /H + exchange system (Webb and Nuccitelli, 1982). This, together with the fact that PKC is involved during Xenopus egg activation (Bement and Capco, 1989), prompted us to measure pHi in Xenopus eggs in response to activation of PKC by phorbol esters and synthetic diacylglycerols. We report that activators of PKC do not produce the activation-associated increase in pHi in Xenopus eggs. In addition, activation by Ca2+dependent activators (pricking or A23187) of eggs pretreated with phorbol esters produced a much smaller increase in pHi than in control non-pretreated eggs. The exocytosis of cortical granules located in the peripheral cytoplasm of Xenopus eggs has long been thought to be Ca2+-mediated, because it can be triggered by pricking the egg cortex only in the presence of Ca 2+ (Wolf, 1974), or by application of the calcium ionophore A23187 (Steinhardt et al. 1974) or by Ca2+ ionophoresis in a specific manner (Cross, 1981). Bement and Capco (1989) have recently reported that PKC activators triggered a complete reaction of cortical granule exocytosis in Xenopus eggs. Intracellular free

Recently, it has been proposed that protein kinase C (PKC) is an integral component of the Xenopus fertilization pathway (Bement and Capco, 1989). Indeed, three activators of PKC, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate (PD) and l-oleoyl-2-acetyl-src-glycerol (OAG), were shown to trigger cortical granule exocytosis and cortical contraction in Xenopus eggs (Bement and Capco, 1989). Activation or fertilization of eggs of the amphibian Xenopus laevis also involves the participation of inositol-l,4,5-trisphosphate [Ins(l,4,5)P3] (Busa et al. 1985) which, like diacylglycerol (DAG), the presumed activator of PKC, is an intermediate produced by the activity of phospholipase C in the phosphoinositide pathway (reviewed by Berridge, 1984). In other systems, such as cultured mammalian cells and sea urchin eggs, PKC activation following cell activation (stimulation with growth factors or fertilization) results in an increase in intracellular pH (pHi) via phosphorylation of a Na + /H + exchanger in the plasma membrane (Burns and Rozengurt, 1983; Swann and Whitaker, 1985). In Xenopus eggs, fertilization is also

Key words: Xenopus, protein kinase C, egg activation, intracellular pH, intracellular free calcium.

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calcium levels ([Ca2+]j) had not been measured under such circumstances. However, Bement and Capco (1990) recently reported the failure of PKC activators to trigger the intracellular Ca 2+ transient rise normally triggered upon egg activation (Busa and Nuccitelli, 1985). In agreement with Bement and Capco's findings, we report that treatment of unactivated eggs of Xenopus with PKC activators does not produce any change in [Ca2+]j. To assess further the role of PKC in Xenopus egg activation, we used inhibitors of PKC, and report that 1 jUM staurosponne or 20 fiM sphingosine did not block A23187- or prick-induced activation. At the higher dose of 100 /AM, a concentration previously reported to inhibit cortical contraction and cortical granule exocytosis in response to A23187 or PMA (Bement and Capco, 1990), sphingosine was surprisingly found to activate a large proportion of the egg populations. Similarly, staurosporine was found to activate the eggs when used at a minimal concentration of 20 /XM. These results are incompatible with the idea that the triggering of egg activation in Xenopus is controlled by the activation or an increase in the activity - of PKC. Our results are compared to those of Bement and Capco (1990), and the role of PKC in Xenopus egg activation is discussed.

Materials and methods Eggs were obtained from Xenopus laevis, reared in the laboratory, stimulated with 800 to 1000i.u. human chorionic gonadotropin (Organon). Eggs were dejellied with 2% cysteine in Fl solution, modified from Hollinger and Corton (1980), which contained (nw): NaCl, 31.2; KC1, 1.8; CaCl2, 1.0; MgCl2, 0.1; NaHCO3, 2.0; NaOH, 1.9, buffered with 10 mM Hepes, pH7.4. Morphological criteria for successful egg activation, detectable under a stereomicroscope, were: (1) the elevation of the vitelline envelope, a consequence of cortical granule exocytosis; (2) the cortical contraction, a transient uniform contraction of the pigmented area (the animal hemisphere) of the egg towards the animal pole; (3) the disappearance of the maturation spot, a consequence of meiosis resumption due to the migration deeper into the cytoplasm of the egg nucleus (evolving from the metaphase II stage of meiosis to the pronucleus stage). Electrical and ionic criteria for successful egg activation, detectable in eggs impaled with microelectrodes (see below), were: (1) the activation potential, a rapid Cl~-dependent plasma membrane depolarization; (2) a transient increase in [Ca2+],; (3) a permanent increase in pHi. With respect to the activation potential, which is the earliest event that can be detected at egg activation, 2 to 5s after pricking the egg cortex or stimulation with the calcium ionophore A23187 (two classical artificial activators of Xenopus eggs), the subsequent events of egg activation could be observed starting at 1-2 min (elevation of the vitelline envelope), 1-5 min (intracellular Ca 2+ transient), 3-4min (cortical contraction), 7-10min (increase in pHi), 20-25min (disappearance of the maturation spot). Each egg, bathed in Fl solution, was impaled with a H + sensitive microelectrode, measuring intracellular pH (pHi) (fabricated as described in Charbonneau et at. 1985) or a calcium-sensitive microelectrode, measuring intracellular free calcium (pCa, the negative logarithm of free Ca2+ activity)

(fabricated as described in Busa, 1986) and with a potential microelectrode measuring the membrane potential. Membrane potential value was subtracted from the pH or calcium microelectrode output at the pen recorder (Linseis) input (Grandin and Charbonneau, 1989a). A complete description of the methods used during electrophysiological recording has been published elsewhere (Grandin and Charbonneau, 1989a). Both neutral carriers used for selective intracellular microelectrodes were from Fluka: hydrogen ion ionophore I (ref. 95291) and calcium ionophore ETH 1001 (ref. 21048). Ca2+ microelectrode and pH microelectrode calibration traces are shown in Fig. 1. The two phorbol esters, PMA (phorbol 12-myristate 13acetate) and PD (phorbol 12,13-didecanoate) and the inactive PMA analogue PDD (4-alpha-phorbol 12,13-didecanoate) were prepared as stock solutions of 1 mM in dimethylsulphoxide (DMSO). The synthetic diacylglycerol SAG (1stearoyl-2-arachidonoyl-src-glycerol) was prepared as a stock solution of Smgml" 1 in ethanol. OAG (l-oleoyl-2-acetyl-57iglycerol), another synthetic diacylglycerol, was prepared as a stock solution of 100 mM in DMSO. The protein kinase C inhibitors, staurosporine and sphingosine, were respectively prepared as stock solutions of 1 and 100 mM in DMSO. All chemicals were purchased from Sigma.

Results

Activation of protein kinase C is not accompanied by any variation in [Ca2+], We examined the effects of an activator of PKC, the phorbol ester phorbol 12-myristate 13-acetate (PMA), on [Ca2+]j and membrane potential of Xenopus laevis eggs. Fig. 2A shows that PMA, although producing two normal events of egg activation, cortical granule exocytosis and cortical contraction (Bement and Capco, 1989), did not induce any change in [Ca2+]j. An absence of an effect of PMA on [Ca2+], in unactivated eggs of Xenopus was observed in all twenty-four experiments. In PKC-stimulated eggs, the extent of cortical granule exocytosis was the same as in normally activated eggs, although exocytosis proceeded at a slower rate (Bement and Capco, 1989). In the case of a normal activation triggered by agents elevating the [Ca2+], (entry of external Ca2+ caused by pricking the egg cortex or intracellular release of Ca from intracellular stores induced by the calcium ionophore A23187), we measured a transient increase in [Ca2+]j (Fig. 2B), in agreement with previous findings (Busa and Nuccitelli, 1985). In addition, PMA did not trigger the activation potential, a Cl~-dependent plasma membrane depolarization, the earliest event that can be detected upon egg activation (Cross and Elinson, 1980). However, PMA was found to have dramatic effects on the membrane potential of unactivated eggs: an initial slow depolarization (peak value around —10 to —2mV) generated within 10 min, followed by a very large hyperpolarization of -30 to — 70 mV, 30-45 min after treatment (Figs 2A, 3A). In those cases in which the membrane potential attained values as large as —50 to -70 mV, spontaneous and rapid depolarizations were frequently recorded. These 'spikes' had a peak potential close to

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that of normal activation potentials, but, unlike the latter, they could occur several times (Fig. 2A). In already activated eggs, PMA did not produce any change in the membrane potential. Activation of protein kinase C does not produce the increase in pHi associated with egg activation In many types of cultured mammalian cells, as well as in sea urchin eggs, a phosphorylating activity of PKC seems to be responsible for the activation of a N a + / H + exchange system (Burns and Rozengurt, 1983; Swann and Whitaker, 1985), which produces the cell activation-associated increase in pHi (Roos and Boron, 1981; Busa and Nuccitelli, 1984). Although Xenopus eggs do not seem to possess a N a + / H + antiporter (Webb and Nuccitelli, 1982), it was interesting to know whether PKC might be implicated in their pHi regulation system. Measurements with intracellular pH microelectrodes demonstrated that the PKC activator PMA did not induce the normal activation-associated increase in pHi (data not shown). Indeed, intracellular pH did not change for at least 90 min following PMA treatment (3/XM), as measured in twelve experiments, although in five other experiments PMA produced a slow decrease in pHi (0.10-0.15 pH unit) (Fig. 3A). Phorbol 12,13-didecanoate (PD), 3 /XM, had no effect on pHi levels in all five experiments performed.

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Fig. 1. Calibration traces of Ca2+ (A) and pH (B) microelectrodes. We chose to show the calibration traces corresponding to the microelectrodes used in the experiments described in Fig. 2A and 3A. (A) Ca2+selective microelectrodes were calibrated in pCa 6.0 and pCa 7.0 solutions, as described in Busa and Nuccitelli (1985), which contained, respectively, 10.0DIM EGTA, 5.0mM CaCl2, lO.OmM Pipes, 45.0mM KOH, 15.0mM KC1, pH6.77 (at 23°C), and lO.OmM EGTA, 5.0mii CaCl2, 10.0mM Mops, 35.5 min KOH, 29.3 mM KC1, pH7.27 (at 23°C). At the beginning of the experiment, this microelectrode had a slope of 33 mV between pCa 6.0 and pCa 7.0. Just after calibration, the solution was replaced by the physiological Fl solution, which is responsible for the large upward deflection in the trace ('Fl'). This is because the Fl solution contains much more free Ca2+ than the pCa 6.0 solution. Eggs were then immersed in Fl solution, in the recording chamber, and impaled, each with a Ca 2+ microelectrode and a potential microelectrode (arrow 'In'). The beginning and end of the recording of [Ca ], in this egg (72 min recording not shown) correspond to the egg recording shown in Fig. 2A. At the end of this experiment, the voltage followers were turned off, the microelectrodes removed from the eggs (arrow 'Out'), and the eggs and Fl solution removed and replaced by pCa 7.0 calibration solution. pCa 7.0 level was exactly the same as at the beginning of the experiment; pCa 6.0 level was slightly different from that at the beginning, giving a slope of 30mV between pCa 7.0 and pCa 6.0, instead of 33 mV at the beginning. In our experiments, we were confronted more by this problem of a small reduction in the microelectrode slope after egg impalement than by the common problem of baseline shift. (B) pH microelectrodes were initially calibrated, as described in Charbonneau et al. (1985), in buffers of pH7.78, 7.27 and 6.77 containing, respectively, 10.0 mM Hepes, 10.0 mM Mops or 10.0 mw Pipes, plus 10.0mM EGTA, 5.0mM CaCl2 and 75.0 mM KC1. However, we noticed that, in some cases, pH microelectrodes displayed signs of instability or difficulty in rapidly attaining a stable level when further immersed in Fl solutions. Therefore, we chose to calibrate our pH microelectrodes directly in Fl solutions at pH6.50, 7.50 and 8.50 (or about these values: the exact values were measured with a Knick 654 pH meter immediately beforehand) buffered, respectively, with 10.0 mM Pipes, 10.0 mM Hepes and 10.0 mM 3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropane sulfonic acid (Ampso). Just after calibration, the eggs were immersed in Fl solution at pH8.50. Achievement of egg impalement was indicated by the large deflection of the pHi trace as the microelectrode entered the egg cytoplasm (arrow 'In'). Three minutes after impalement, the Fl solution at pH8.50 around the eggs was replaced by Fl solution at pH7.5O (arrowhead), which is marked on the trace by the slight acidification of the egg cytoplasm. The beginning and the end of recording of pHi level in this egg (114 min recording not shown) correspond to the trace shown in Fig. 3A. At the end of the experiment, the voltage followers were turned off, the microelectrodes pulled out of the eggs (arrow 'Out'), and the Fl solution in the recording chamber was replaced by a fresh solution that had been adjusted at pH7.50 immediately beforehand.

Pretreatments with phorbol esters diminish the pHi and [Ca2+]i responses to egg activation As previously reported, phorbol esters do not lead to

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Charbonneau triggering the [Ca 2+ ]j and pHi increases, or the activation potential, as seen above. Could these ionic events be triggered by mobilization of intracellular Ca 2 + stores following partial activation with PKC activators? Eggs pretreated with 3 /IM PMA or 3 /IM PD displayed an increase in pHi when further challenged with Ca 2+ -dependent activating signals (Fig. 3A). However, the activation-associated increase in pHi in eggs pretreated with 3 /XM PMA for at least 30 min and stimulated with 0.5 or Ipm A23187 was much smaller, 0.09±0.04pH unit (S.D., n=14), than in control eggs (Table 1). The kinetics of the A23187-induced increase in pHi in eggs pretreated with phorbol esters suggested that these changes were physiological. In order to rule out the possibility of an artefactual change in pHi in phorbol ester-pretreated eggs, due to the Ca / H + exchange properties of A23187, Xenopus eggs were impaled with pH microelectrodes, activated by pricking, and, once pHi had attained a stable elevated value (around 40min after egg activation), treated with 2JIM A23187. Under such conditions, A23187 produced, around 10min after addition, only a slight change in pHi, a 0.02 to 0.04 pH unit cytoplasmic acidification, as measured in seven eggs.

6.5-

Fig. 2. (A) Effect of the phorbol ester PMA (phorbol 12myristate 13-acetate) on the membrane potential (Em) and intracellular free calcium activity (pCa) of an unactivated Xenopus egg. The egg, bathed in Fl solution, was impaled with a potential microelectrode, measuring membrane potential (top trace) and a calcium-selective microelectrode, measuring the activity of intracellular free calcium (bottom trace). Addition of 3 /.m PMA (arrow 'PMA') induced, within a few minutes, a small depolarization, followed by a dramatic hyperpolarization from — 12 to -73 mV with, at intervals, appearance of dramatic transient depolarizations. However, PMA did not induce any changes in the intracellular free calcium level, which remained at pCa 6.42 (0.38 /JM). The egg was rinsed with Fl solution (arrow 'Fl') 30min after PMA addition to avoid possible lysis. All measurements performed in the study on the effects of phorbol esters indicate, in unactivated eggs, a basal [Ca2+]i of 0.37±0.11^M (S.D., n=37) and a membrane potential, measured in eggs impaled with two microelectrodes (a potential microelectrode and a calcium or pH microelectrode), of -11.0±2.2mV (S.D., n=93). (B) Representative changes in membrane potential (top trace) and intracellular free calcium (bottom trace) following activation in an egg of Xenopus. The egg, impaled with a potential microelectrode and a calcium-selective microelectrode, was bathed in Fl solution containing 3/an of the inactive PMA analogue, PDD (4-alpha-phorbol 12,13-didecanoate). The egg was activated by pricking (arrow 'Pricking'), 45 min after PDD addition. A transient increase in intracellular free calcium concentration was detected 5min after egg activation, indicated by the occurrence of the activation potential (arrow 'AP').

cell cycle progression in Xenopus eggs, and are therefore partial activators (Bement and Capco, 1989). In particular, PKC activators are not capable of

In addition, an intracellular Ca 2 + transient was recorded in only three out of fourteen experiments in which eggs were pretreated with 3 [MM PMA for at least 30min and stimulated with 0.5 or 1 fXM A23187. In contrast, an intracellular Ca 2+ transient was recorded in all nine control experiments (no pretreatment). In contrast to the pHi and intracellular Ca 2 + responses to egg activation, which were modified after pretreatment with phorbol esters, as seen above, the activation potential remained similar to that in controls. Indeed, the peak value of the activation potential after stimulation with 0.5 or I ^ M A23187 was found to be around+5mV in control non-pretreated and PDDtreated eggs, as well as in PMA- and PD-treated eggs (Table 1). Since the only known mode of action of artificial activators of Xenopus eggs, such as A23187, is to release Ca 2 + intracellularly, it seemed reasonable to assume that A23187 did produce an intracellular Ca 2 + transient in PMA-pretreated eggs. However, in such eggs, that Ca 2 + transient would not have been detected by the Ca 2 + microelectrodes, possibly due to the fact that it was smaller than that in non-pretreated eggs (see Discussion). In order to evaluate such a possibility, PMA-pretreated eggs (3/IM, 20-30 min) were microinjected with 100 ITIM BAPTA, a potent chelator of Ca 2 + ions, and further challenged with I ^ M A23187. Under these conditions, the eggs did not activate, that is did not display the activation potential, as observed in all eight experiments performed (Fig. 4). Although the eggs reasonably supported the microinjection of BAPTA, the subsequent addition of A23187 rapidly produced their complete lysis. The period of time between A23187 addition and egg lysis, sufficient to assess the absence of an activation potential in PMApretreated eggs microinjected with BAPTA, was not long enough to record pHi until the moment corresponding to the physiological changes (7-10 min after

Protein kinase C and egg activation

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Fig. 3. (A) Changes in intracellular pH in an egg of Xenopus following treatment with phorbol 12-myristate 13acetate (PMA) and activation with the calcium ionophore A23187. The egg, bathed in Fl solution, was impaled (20min before the beginning of the trace shown here) with a potential microelectrode, measuring the membrane potential (E m ; top trace), and a pH-selective microelectrode, measuring the intracellular pH A231S7 F1 (pFIi; bottom trace). Addition of 3 tm PMA (arrow 'PMA') induced a slow cytoplasmic 10rB acidification, from pH7.47 to pH7.38, and a large hyperpolarization of the membrane. The egg was activated with 1 /AM A23187 -20(arrow 'A23187'), 35min after 10min PMA addition, which generated a large activation potential (arrow 'AP'), from the potential level attained after PMA treatment 7.81(-40 mV) to the equilibrium potential for chloride ions (+2mV). Activation also triggered a transient acidification followed by a small alkalization, from pH7.38 to pH7.53 (see mean values in Table 1). The egg was rinsed with Fl solution (arrow 'Fl') a few minutes after addition of A23187. Rinsing is probably responsible for the re-depolarization following the rapid hyperpolarization which occurred soon after the plateau value of the AP had been attained. Indeed, that large hyperpolarization, abnormal for an activation potential, was probably due to the presence of PMA in the recording chamber. Removal of PMA upon rinsing brought the membrane potential back to a less negative value, typical of a normal AP. In the whole study on the effects of phorbol esters, pHi in unactivated eggs, before treatment, was 7.48±0.07 (S.D., n=35). (B) Normal changes in intracellular pH following activation of a Xenopus egg. The egg, impaled with a potential microelectrode and a pH-selective microelectrode, was incubated for 30min in Fl solution containing 3 fiM of the inactive PMA analogue, PDD (4-alphaphorbol 12,13-didecanoate). Activation, induced by IJIM A23187 (arrow 'A23187'), triggered a typical activation potential from -12 to +3mV (arrow 'AP'; top trace) and, following a small transient acidification, a large alkalization of the egg cytoplasm, from pH7.45 to pH7.77 (see mean values in Table 1).

the activation potential). Thus, the A23187-induced changes in PMA-treated eggs (at least the activation potential) can be abolished by microinjection of a Ca2+ chelator. Meanwhile, in the same recording chamber, eggs that had also been pretreated with PMA but not microinjected with BAPTA, displayed a normal activation potential in response to 1 ^M A23187 in all eight experiments in which the neighbouring BAPTAinjected eggs did not activate (Fig. 4). Absence of effects of synthetic diacylglycerols on pHi and [Ca2+]i in Xenopus eggs To extend our findings concerning the relationships between PKC activation and [Ca2+]j and pHi changes, we also used two synthetic diacylglycerols: l-oleoyl-2-acetyl-srt-glycerol (OAG) and l-stearoyl-2-arachidonoyl-srt-glycerol (SAG). Both OAG and SAG have previously been shown to activate PKC (Bell, 1986). In addition, OAG stimulates cortical granule exocytosis in Xenopus eggs (Bement and Capco, 1989). Neither OAG, at concentrations up to

100/XM, nor SAG, at concentrations as high as 77/iM

(SO^gml"1), produced any change in pHi (six experiments with OAG, five with SAG) or [Ca2+]j (eight experiments with OAG, thirteen with SAG) in unactivated eggs of Xenopus. In addition, eggs pretreated with 100JIM OAG or 77/JM SAG, for at least 45min,

remained perfectly activatable by A23187, and generated an intracellular Ca 2+ transient (five-experiments with OAG, eight with SAG) and an increase in pHi (eight experiments with OAG, four with SAG) identical to those in non-pretreated eggs. Thus, synthetic diacylglycerols seem to be less potent than PKC activators in affecting the pHi and intracellular Ca 2+ responses to Ca2+-mobilizing agents. Effects of PKC inhibitors on Xenopus egg activation We used staurosporine and sphingosine which, although not entirely specific, are potent inhibitors of PKC (Davis et al. 1989; Huang, 1989). Treatment of unactivated eggs for at least 2 h with 1 /XM staurosporine or 20/JM sphingosine had no effect on egg activation.

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Table 1. Effects of phorbol esters on the egg activation response (pHi level and activation potential) to Ca2+-dependent activating signals in Xenopus laevis a Treatment PMAb PD

b

PDD

b

non-pretreated eggs

Activation potential peak value0 (mV)

pHi increased (pH unit)

+4.3±2.3 ("=27) +5.2±1.3 ("=5) +4.0±1.7 ("=14) +4.9±1.7 ("=25)

0.09±0.04 ("=14) 0.22±0.09 («=5) 0.31±0.03 («=4) 0.31+0.04 ("=9)

"Results are expressed as mean values±Standard Deviations (number of experiments). b The two phorbol esters, PMA (phorbol 12-myristate 13acetate) and PD (phorbol 12,13-didecanoate), and the inactive PMA analogue PDD (4 alpha-phorbol 12,13-didecanoate) were used at a final concentration of 3/iM. Treatment of unactivated dejellied eggs of Xenopus was for 30 to 45 min. Then, the eggs were stimulated with the calcium lonophore A23187 (0.5 or 1/^M) c Equilibrium potential attained at the plateau of the egg activation-induced activation potential (Cl~-dependent plasma membrane depolarization). d Amplitude of the egg activation-induced pHi increase measured at the plateau level (30 to 40 min after egg activation).

Indeed, in response to either pricking or A23187 (0.5 UM), events characteristic of egg activation, such as the activation potential, cortical contraction, elevation of the vitelline envelope, pHi rise and meiosis resumption were triggered in eggs pretreated with either of the PKC inhibitors used. Bement and Capco (1990) recently reported an inhibition of cortical contraction and cortical granule exocytosis, in response to A23187 or PMA, in eggs pretreated with 100 ^M sphingosine. Our results are somewhat different. Indeed, we noted that 100/iM sphingosine alone activated the majority of the eggs (75-90% in a mixture of eggs from two or three females). In some females (taken individually), 100^M sphingosine was even found to activate 100 % of the eggs. The activating effect of 100//M sphingosine was observed in all four experiments performed, using eggs from nine females (at least thirty eggs for each experiment). Activation by 100(JM sphingosine was evident morphologically, under a stereomicroscope: elevation of the vitelline envelope, cortical contraction and disappearance of the maturation spot. Morphologically, the first signs of sphingosine-induced egg activation were evident as early as 5 min after treatment. The majority of the egg population was activated 10-20 min after treatment with sphingosine, whereas the last eggs were activated around 40 min after treatment with sphingosine. Activation of Xenopus eggs by 100 fm sphingosine was confirmed at the electrical and ionic levels: the occurrence of an activation potential (Fig. 5), intracellular Ca2+ transient (Fig. 5A) and activation-associated increase in pHi (Fig. 5B) within 10-30 min of sphingosine addition. To know whether the ability of sphingosine to activate

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Fig. 4. Effects of the microinjection of BAPTA (l,2-bis(2aminophenoxy)ethane-A',Af,7V',A''-tetraacetic add), a highly selective calcium chelating reagent, on the activation response to A23187 in PMA-pretreated eggs. Eggs were first incubated in Fl solution containing 3 im of the phorbol ester PMA (phorbol 12-myristate 13-acetate), impaled each with a pH microelectrode and a potential microelectrode (see Fig. 3), and, 20-30 min after beginning of the treatment, were microinjected with 30—40 nl of 100 iriM BAPTA (prepared in 10CTLMHepes buffer and adjusted to pH7.5 with NaOH) (arrow 'BAPTA'). In each experiment, two eggs were impaled with microelectrodes in the same recording chamber, but only one of the two was microinjected with BAPTA (Em and pHi top traces), the second egg serving as a control (Em and pFfi bottom traces). Around 5 min after microinjection of BAPTA, the eggs were stimulated with A23187 (1 ^M final concentration) (arrow 'A23187'). A23187 triggered a typical activation potential (arrow 'AP') in the non-injected egg, whereas the egg previously microinjected with BAPTA did not display the activation potential. This suggests that in PMA-pretreated eggs some intracellular Ca release (that can be blocked by BAPTA microinjection) must take place in order to trigger the activation potential. An absence of activation potential in the PMA-pretreated egg microinjected with BAPTA and stimulated with A23187 in association with the occurrence of an activation potential in the control egg placed in the same recording chamber but not microinjected with BAPTA, was observed in all eight experiments performed.

Xenopus eggs was shared with other inhibitors of PKC, we studied the effects of staurosporine at concentrations higher than 1 JIM, an ineffective concentration, as seen above. Interestingly, staurosporine also triggered activation of Xenopus eggs when its concentration in the surrounding medium was at least 20/XM. Morphologically, staurosporine-induced egg activation became evident in about 50 % of the egg population around 15 min after treatment: elevation of the vitelline envelope and disappearance of the maturation spot. The maximal percentage of egg activation (80-100%) induced by 20 JIM staurosporine was achieved around

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7.9

Fig. 5. Effects of 100/iM sphingosine on unactivated eggs in Xenopus laevis. (A) In a large majority of cases, sphingosine, at 100 ^M, was found to activate the eggs, as illustrated here. At the arrowhead 'Sphingosine', the Fl solution around the impaled eggs in the recording chamber was exchanged for an Fl solution containing 100/iM sphingosine. After a 30min delay, sphingosine triggered an activation potential (arrowhead 'AP'; peak value: — 2mV), followed by a typical intracellular Ca transient (from 0.45 /*M to 0.93 /IM). (B) Sphingosine, 20min after addition (arrowhead 'Sphingosine'), also triggered a typical activation-associated increase in pHi (from pH7.48 to pH7.82). This egg was from a different female from that used in the experiment shown in A. In both A and B, egg activation, followed under a stereomicroscope during recording, was also evident morphologically (see text).

50min after treatment. The activating effect of 20 ^M staurosporine was observed in all three experiments performed, using eggs from seven females. Treatment with 20 HM staurosporine also resulted in the occurrence of an activation potential, a typical intracellular Ca2+ transient and a typical increase in pHi (data not shown). Finally, it should be noted that in eggs activated by 20 fJM staurosporine, two of the events of egg activation differed from those present in classically activated eggs (by sperm, A23187 or pricking). First., the elevation of the vitelline envelope occurred very progressively, reaching its maximal extent around 20min after the time it was first detected under the stereomicroscope. In normally activated eggs, that reaction is complete around 5min after its initiation. Second, there was no cortical contraction in eggs activated by 20 /XM staurosporine. Discussion

The present study demonstrates that activation of protein kinase C (PKC) by phorbol esters does not produce the physiological changes in [Ca2+]j and pHi

467

associated with activation oiXenopus eggs. This implies that cortical granule exocytosis, which can be triggered by phorbol esters in Xenopus eggs (Bement and Capco, 1989), can occur without noticeable variation in [Ca ],. Moreover, the absence of [Ca2+]; and pFIi variations during phorbol-ester-induced activation correlates well with the observations that phorbol esters cannot trigger meiosis resumption in Xenopus eggs (Bement and Capco, 1989) and that protein kinase C appears to act downstream of the egg-activation-associated [Ca2+]; rise to trigger cortical granule exocytosis (Bement and Capco, 1990). In contrast, our results with sphingosine and staurosporine, two inhibitors of PKC, suggest that the release of the metaphase block might be correlated with a decrease in PKC activity. Absence of an effect of PKC activators on pHi in Xenopus eggs can be correlated with an absence of Na+/H+ exchange system On first analysis, the absence of an effect of phorbol esters on the intracellular pH of unactivated eggs of Xenopus can be explained by the fact that these eggs lack a Na + /H + exchange system (Webb and Nuccitelli, 1982; Grandin and Charbonneau, 19896). Indeed, in other systems, phorbol esters or synthetic diacylglycerols activate a Na + /H + exchanger with a resultant pHi rise, for example in sea urchin eggs (Swann and Whitaker, 1985; Lau et al. 1986; Shen and Burgart, 1986) and cultured mammalian cells (Burns and Rozengurt, 1983; Besterman and Cuatrecasas, 1984; Moolenar et al. 1984). Since phorbol esters and synthetic diacylglycerols presumably activate PKC specifically (Nishizuka, 1984, 1986, 1988), it appears that the Na + /H + exchanger is directly turned on by this kinase. The egg of Xenopus appears to represent a particular system in which PKC, although involved in the activation pathway as recently demonstrated (Bement and Capco, 1989), does not seem to be responsible for the activation-associated pHi rise, as shown in the present study. Phorbol-ester-induced cortical granule exocytosis occurs in the absence of [Ca2+]l variations Our results demonstrate that the phorbol ester PMA, previously shown to trigger a complete exocytosis of the cortical granules in Xenopus eggs (Bement and Capco, 1989), does not induce any variation of [Ca 2+ ] i; thus confirming a recent report (Bement and Capco, 1990). This has already been described in human neutrophils, in which PMA triggers exocytosis of secondary granules even when [Ca2+]j is lowered 10-20 times below the normal resting level (Di Virgilio et al. 1984). This was later confirmed in several systems (see references in Knight et al. 1989) and interpreted as resulting from a phorbol-ester-induced increase in the sensitivity of the granules to [Ca2+], (Knight et al. 1989). In other words, the Ca2+-independent exocytosis observed in several systems appears to be due to an increased sensitivity of the secretory process to intracellular Ca 2+ rather than to a Ca2+-independent pathway (Knight et al. 1989). The observations by Bement and Capco (1989) that

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subthreshold concentrations of PMA and A23187 act synergistically to trigger exocytosis in Xenopus eggs, together with the present finding and that by Bement and Capco (1990) that PMA triggers exocytosis without [Ca 2+ ] ; variation, suggest that cortical granule exocytosis in Xenopus eggs represents one of these so-called types of Ca2+-independent exocytosis, which is, in fact, a Ca2+-dependent exocytosis in which activation of PKC increases the sensitivity of the secretory granules to [Ca2+]i. A role for protein kinase C in Xenopus egg activation So far, the only evidence for a precise role of PKC in Xenopus egg activation is that provided by the recent work of Bement and Capco (1989), who have demonstrated that the two phorbol esters PMA and PD and the synthetic diacylglycerol OAG trigger cortical granule exocytosis and cortical contraction. Using the same activators of PKC, at the same concentrations (3,UM for PMA and PD, 100JJM for OAG), we have demonstrated that PKC did not appear to be involved in the physiological [Ca2+]; and pHi changes accompanying activation of Xenopus eggs. In addition, we have demonstrated that two potent inhibitors of PKC, staurosporine and sphingosine, at 1 and 20 fiM, respectively, did not interfere with any event of egg activation. Yet, phorbol esters, principally PMA, seem to have dramatic effects on the membrane potential of Xenopus eggs, but not of the kind that are triggered during normal egg activation. These changes in membrane potential might be related to the previously described modulation, in various cell types, of ion conductance by PKC, via phosphorylation of ion channel proteins, ion exchange proteins or pumps (reviewed by Nishizuka, 1986). However, in Xenopus eggs, it cannot be excluded that PMA might act on some ion channel conductance independently of its effect as an activator of PKC, as has been recently proposed concerning the depression of Ca2+ current in chick sensory neurones by the phorbol esters Otetradecanoylphorbol-13-acetate and phorbol-12,13-diacetate (Hockberger et al. 1989). Although activation of PKC and the associated cortical granule exocytosis in Xenopus eggs do not appear to be coupled to pHi or [Ca2+]i changes, as seen above, it is interesting to note that pretreatment with one of the two phorbol esters used, PMA, but not with the synthetic diacylglycerols OAG and SAG, prevented the intracellular Ca transient and strongly reduced the increase in pHi following stimulation with A23187 (see Table 1). The kinetics of the reduced increase in pHi in PMA-pretreated eggs stimulated with A23187, identical to those in normally activated eggs, as well as our experiments on the effects of A23187 on pHi of already activated eggs, demonstrate that the increase in pHi in PMA-pretreated eggs is not artefactual. During normal egg activation, the intracellular Ca 2+ transient precedes the increase in pHi by 5 to lOmin. Presumably, the increase in pHi is a consequence of the intracellular Ca 2+ transient, because the intracellular Ca 2+ transient is believed to represent some sort of

triggering signal during egg activation. How can we explain the occurrence, in PMA-pretreated eggs stimulated with A23187, of an increase in pHi, even with a reduced amplitude, in the absence of an intracellular Ca2+ transient? It might well be possible that, in such eggs, [Ca2+]i did change following stimulation with A23187, but in an altered way, perhaps because the change did not proceed as a wave as is normally the case (Busa and Nuccitelli, 1985). We believe that [Ca2+]; changes in Xenopus eggs are more reliably detected with the Ca2+ microelectrodes when they correspond to a massive propagating front, whereas smaller [Ca2+]j changes occurring simultaneously in various distinct regions of the cytoplasm might be missed by the Ca2+ microelectrode tip. We propose that, in PMA-pretreated eggs, A23187 would produce more discrete [Ca2+], changes than those taking place during normal egg activation. These smaller [Ca ], variations, which, in addition, might not be propagating as a wave, might in turn result in a reduced increase in pHi. The assumption that A23187'did produce [Ca2+]j variations in PMA-pretreated eggs is confirmed by the observation that microinjection of BAPTA, a Ca2+ chelator, into such eggs could prevent the occurrence of the activation potential in response to A23187. This demonstrates that some intracellular Ca2+ must be released into the egg cytoplasm, although it cannot be detected with the Ca microelectrode for reasons explained above, in order to trigger the activation potential. In contrast to the results of Bement and Capco (1990), who recently reported a blockade of A23187 or PMA action by 100/iM sphingosine, we observed a direct activation of a very large proportion of eggs by 100 [M sphingosine. Sphingosine competes with the natural diacylglycerol on the regulatory domain of PKC (reviewed by Huang, 1989). Sphingosine, although not entirely specific, is known to be a potent inhibitor of PKC (Davis et al. 1989; Huang, 1989). The situation found here, in which sphingosine (100 ^M) activates Xenopus eggs, is very similar to that recently described in Xenopus oocytes in which sphingosine (170 ^M in the incubation experiments) induced oocyte maturation (Varnold and Smith, 1990). The activating effect of an inhibitor of PKC on Xenopus eggs is confirmed, in the present study, by the utilization of staurosporine (20 ^M), another potent inhibitor of PKC. A straightforward explanation of the present finding, that inhibition of PKC triggers egg activation in Xenopus, is that a decrease in PKC activity is required for egg activation, as appears to be the case for oocyte maturation (Varnold and Smith, 1990). Both events, oocyte maturation and egg activation, correspond to the release of a meiotic block, in prophase and metaphase, respectively. However, it should be kept in mind that at these very high concentrations, 20 and 100 /JM, respectively, staurosporine and sphingosine might have effects unrelated to PKC. It is therefore premature to conclude that a decrease in PKC activity is associated with - or required for - Xenopus egg activation. In addition, our observation that sphingosine and staurosporine, two

Protein kinase C and egg activation

inhibitors of PKC, activate the eggs of Xenopus might explain our other observation that the pHi and [Ca ] ; responses to Ca2+-dependent activating signals are diminished when eggs are pretreated with activators of PKC (Fig. 3; Table 1). Indeed, if a decrease in PKC activity is required during egg activation, then it is logical that activation of PKC prior to application of activating stimuli might interfere with the ionic responses to egg activation. We cannot explain why our results with 100 ^IM sphingosine are totally opposite to those of Bement and Capco (1990), who reported an inhibition of A23187-induced egg activation by the same drug at the same concentration. Recent experiments on the effects of various agonists and antagonists of PKC on Xenopus oocyte maturation have also led to quite variable, and sometimes opposing, results (see references in Varnold and Smith, 1990). In the case of Xenopus egg activation, inhibition of PKC seems to be involved in the resumption of the cell cycle (release of the metaphase block), as shown here, while, on the other hand, activation of PKC appears to be required for cortical granule exocytosis. This is not a unique situation, since in hamster eggs the response to GTP[S], which generates diacylglycerol and activates PKC, is inhibited by the PKC agonists phorbol 12-myristate 13acetate and diC8 (1,2-dioctanoyl-glycerol) and enhanced by the PKC inhibitor sphingosine, suggesting the existence of a feedback inhibition of that response by PKC (Swann etal. 1989). Future experiments on the role of PKC in Xenopus egg activation should be directed at examining the kinetics of changes in the activities of the natural diacylglycerol DAG and of PKC, associated with egg activation.

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R. (1985). External N a + inhibits Ca 2+ -ionophore activation of Xenopus eggs. Devi Biol. 108, 369-376. CROSS, N. L. (1981). Initiation of the activation potential by an increase in intracellular calcium in eggs of the frog, Rana pipiens. Devi Biol. 85, 380-384. CROSS, N. L. AND EUNSON, R. P. (1980). A fast block to polyspermy in frogs mediated by changes in the membrane potential. Devi Biol. 75, 187-198. DAVIS, P. D . , H I L L , C. H., KEECH, E., LAWTON, G., NIXON, J. S., SEDGWICK, A. D . , WADSWORTH, J., WESTMACOTT, D. AND

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We thank Dr Ray Kado (Gif-sur-Yvette, France) for the design of the electronic recording system, and Mr Jean-Paul Rolland for building it. This work was supported by grants from the Ligue Nationale contre le Cancer (Comite' D6partemental d'llle-et-Vilaine), Fondation pour la Recherche Me'dicale, Fondation Langlois, Association pour la Recherche sur le Cancer and R6gion Bretagne. NG was a recipient of a doctoral fellowship from the Region Bretagne.

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