The four dimensions of calcium signalling in Xenopus oocytes

Cell C&dun

(Wl)

12, 217-227

Q Langmen Group UK Ltd 1991

The four dimensions of calcium signalling in Xenopus oocytes S. DELISLE Pulmonary Division, Department of Internal Medicine, University of lowa College of Medicine, Iowa City, Iowa, USA

Abstract -

This review focuses on the inositol phosphate/Ca** signalllng pathway in

Xenupus oocytes. The known characteristicsof the individual elements of this cascade -

from the membrane receptors to the intracellular Ca*’ stores - will be covered. Based on this knowledge, a simple model will then try to account for the behaviour of the newly recognized oscillations of free intracellularCa*+ and propagatedCa*+ waves. Finally, some of the potential physiological functions of the inositol phosphate pathway will be summarized. Although there is no systematicattempt to contrastthe findings in the oocyte to those in other cells, the readers of this journal will not fail to notice a high degree of similarity. Although this may seem unexciting at first, it suggests that the inositol phosphate signalling pathway may be strikingly conservedacross species. Measuring intraceiiuiar Ca2’ in Xenupus oncytes Females of the South African clawed toad Xenopus luevis harbor hundreds of oocytes in their ovaries. The mature oocytes (Dumont’s stage V and VIUI) are large (1 - 1.3 mm) and robust cells that are readily amenable to electrophysiological studies. When voltage-clamped oocytes are suddenly depolarized from their resting membrane potential (-40 to -60 mV [2]) to voltages mom positive than -30 mV, a transient current carried by Cl- ions develops [3, 41. This current can be blocked either by preinjecting the oocyte with EGTA [51 or by substituting extracellular calcium (Ca2’,> with S12’ or Mg2+ [4]. Thus, it requires the entry of Ca2+, into the cell. A Cl- current can also be created by 2+ Abbreviations: Ca’+, calcium; Ca O, extracellular calcium; [Ca2+]b free Mrscellular calcium concctltion; inositol phosphate; InsP, inositd Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; Ins (1,3,4,5P4, 1,3,4,%etrakisphosphate.

injecting Ca2+ intracellularly [5-71 or releasing Ca2+ horn Ins(l,4,5)P3-sensitive stores [6-121. Thus, the oocyte possesses endogenous Ca2’-sated Clchannels - patch-clamp discloses small (slope conductance = 3 pS) chloride-selective channels directly activated by Ca2’ [13], but there may be more than one channel type [2, 141. Because of these Cl- channels, a rise of free intracellular Ca2’ concentration ([Ca2+]i) causes Cl- currents which am easily measured by two-electrode voltage-clamp. Although most convenient, this assay has the following limitations: first, it probably represents [Ca2+]i changes occurring just beneath the cytoplasmic membrane [6 151; second, it integrates all submembranous [&Ii changes across the oocyte surface and thus does not contain spatial information; third, it only gives relative changes in [Ca2+]i, not absolute values. Because of these limitations, the Ca2’-gated Cl- current assay has often been supplemented by Ca2’-sensitive electrodes [16-181 and fluorescent Ca2+ indicators [7, 13, 19-221.

217

218

Inositol phosphate second messengerpathway Membrane receptor

cELLcxL4mM

This promotes receptor-(GDP-a-&) binding catalyses the replacement of GDP by Mg2 -GTp, (b) The low affinity GTP-a-@ complex dissociates from the receptor and the activated GTP-a monomer separates from the &Jcomplex; (c) GTP-cl stimulates an effector (in the case of the inositol phosphate pathway, a candidate for this effector is phospholipase C); (d) GTP-a hydrolyses GTP to GDP and terminates the signal. Because this last step requires several seconds to complete, GTP-a accumulates causing signal amplification. In the oocyte, ACh and serotonin responses are inhibited by guanosine 5’-0-(2-thio)bisphosphate (GDP@ [351or pertussis toxin @‘TX)[32, 35, 511. In addition, injection of guanosine 5’-O-(3-thio)triphosphate (GTP@) releases Ca2’ from Ins(l,4,5)Ps_sensitive stores [32, 35, 521 and PTX catalyses the ADP-ribosylation of a 40 kD protein [32, 51, 521. Together, these data suggest that a PTX-sensitive G-protein links cell membrane receptors to the inositol phosphate pathway. Recently, cDNA 89% homologous to the rat Go a-subunit has been cloned from the oocyte cDNA library [53]. Injection of the activated Go a-subunit releases Ca2’ from intracellular stores whereas three different Gi a-subunits do not [54]. Also, preinjection with holo-Go increases the oocyte response to ACh [54]. Thus, Go-proteins can translate a signal from a membrane receptor to the InsP pathway.

Stimulation of the oocyte with acetylcholine (ACh) causes a Cl- current [23] and, therefore, a rise in [Ca2+]i 1241. Oron et al. found that injecting inositol 1,4,Qrisphosphate (Ins(1,4,5)P3) reproduced the Ach response [25]. In addition, 3H-inositol-labelled oocytes stimulated with ACh accumulated phosphatidylinositol 4,5-bisphosphate (FW2) breakdown products such as inositol l+bisphosphate (InS(l,d)P2) and InsP3. This was the first direct evidence physiologically linking the ACh receptor to inositol phosphate (InsPa) metabolism. Since then, both endogenous and mRNA-expressed receptors coupled to PlP2 hydrolysis have been described - these include ACh [6, 10, 15, 24-291, serotonin [13, 30-351, glutamate [361, substance K [21, 371, neurotensin [38], thyrotropin-releasing hormone (TREI) [39], a-thrombin [40], gonadotropin-releasing hormone [21,411, fMet-Leu-Phe peptide [42], dopamine [43], angiotensin II [21], and a receptor to an undefined 60-70 kD serum protein [44]. When a cytoplasmic membrane receptor is repeatedly stimulated, the magnitude of its Cl’current response decreases [31, 32, 3436, 38, 45, 461. This ‘desensitization’ also occurs when a cell is repeatedly injected with Ins(1,4,5)P3 [7, 10, 471. Although there is also some desensitization of the Ca2+-gated Cl- channels themselves [4, 48, 491, preinjecting an oocyte with Ca2’ does not attenuate its subsequent response to serotonin [34]. Thus, most of the desensitization occurs distal to the InsPs Ins(l ,IJ)Ps-induced Ca2’ signal generation but proximal to the Ca2’-gated Clchannels. CB’ stores The internal organelles of a large cell can be G-proteins separated by centrifugation while leaving the integrity of the cytoplasmic membrane intact [55, G-proteins are Mg2’/GTP activated heterotrimers 561. Using such stratified Xenopus eggs, Han and (a, fl and y subunits) that link membrane receptors Nuccitelli demonstrated that the endoplasmic to endogenous second messenger pathways. They reticulum (RR) layer contains virtually all of the have been broadly divided into GS(slimulatory), Gl Ins(l,4,5)Ps-releasable C!a2’ 1161. Of course, the (inhibitory) and Go (others). For the purpose of this possibility that the InsPs-sensitive Ca2’ pool lies in discussion, their proposed mechanisms of action can a distinct RR-like organelle, such as the be summarized as follows (for details, see [50]): ‘calciosome’ [57], has not been ruled out Although (a) The binding of an agonist to its receptor the intracellular distribution of the In@-sensitive

XENOPUS OOCYTES : THE 4 DIMENSIONS OF Ca SIGNALLING

stores is not yet known, at least some stores must be located close to the cytoplasmic membrane [58-601. The animal (pigmented) hemisphere of the cell discloses a higher sensitivity to ACh [61, 621 and Ins(1,4,5)Ps [9, 10, 44, 47, 60 stimulation. Because 1+ intracellular injections of Ca also induced larger Cl- currents at the animal pole than at the vegetal pole [5], the simplest explanation for this asymmetry is a greater density of Ca2+-gated Cl- channels at the animal versus vegetal hemisphere. However, the possibility of an uneven distribution of ACh receptors or of Ins(l,4,5)P3_sensitive Ca2+ pools As mentioned, there is a rapid remains. desensitization to repeated injections of Ins(1,4,5)P3 [7, 10, 471. However, Berridge 1471 could desensitize the InsPs response at a given cell locus without affecting the response to InsP3 injected 200 pm away. several InsP3-sensiZZ”Ca’stZZtetha~Lr YZZ Ca2+ independently from each other (this is in keeping with observations in pancreatic cells [63 ). 1 The oocyte also contains an InsPa-sensitive Ca + store (P.M. Snyder, personal communication & [64]). The evidence that Ca2’ within this store can be released by an elevation of [Ca2+]i remains inconclusive [ 15, 241. Thus, the search for the physiological regulator of the InsPs-sensitive Ca2’ store(s) continues. Ins(l,4,.5)P&uluced Cg’ release In the absence of extracellular Ca2’, an injection of Ins( 1,4,5)P3 tri gers a brief release of stored 2f intracellular Ca (0.5-2 min) [7, 8, 10, 251. This is followed by a prolonged (8-9 min) and oscillatory release of Ca2’ [6-8, 15, 341. While the mechanism of the second Ca2’ release temains speculative [34], a review of the properties of the initial release is warranted. Ca2+-gated Cl- currents do not occur until a certain amount of Ins(1,4,5)Pa is injected [7, 20, 60, 651. This ‘threshold’ probably occurs because a minimum amount of Ins(1,4,5)Pa is required to release stored Ca2’ , and not because a certain Ca2+-gated to activate [Ca2+]i is needed Cl--channels [60]. The time required to reach this threshold may explain the delay observed between agonist stimulation and the onset of Ca2’ release [60, 66, 671. As would be expected, this latency

219

time shortens at higher concentrations of agonist [7, 20, 601. Ins(l,4,5)Ps_induced Ca2+ release has two additional characteristics: (a) It is a highly co-operative process (Hill’s coefficient = 3.7). This co-operativity provided for signal amplification as small changes in Ins(1,4,5)Ps concentration can release large amounts of Ca2+ WI. (b) It is inhibited by a rise in [Ca2’]i [7, 111. The presence of co-operativity and feedback inhibition by Ca2’ is not unique to the oocyte Ins(1,4,5)Ps response (see [67, 681 and references therein). Ins( 1,4,5)P3&imulation raises [Ca2+]i from its resting level of 50-90 nM [21, 691 to 0.25-l pM [16,21] (Dascal [2] discusses the variability of these measurements). Ca2+ ions released from InsPa-sensitive stores (perhaps in exchange for Nat [70]) may then follow different fates. First Ca2’ may be taken back into the ER ;ia a vanadate-inhibitable Ca2’-ATPase [16, 711. A fast phase (10 s) removes 80% of the Ca2+, while a subsequent slower phase (60 s) removes the rest (this dual kinetic is not unlike other Ca2+-ATPases, albeit faster 172-761). Second, the cell probably extrudes Ca2+ as suggested by the presence of 45Ca2’ efflux in stimulated oocytes [24, 39,77,78]. The relative contribution of these two pathways in limiting the rise of [Ca2+]i is not known at present. Influx of extracellular C2’ In the presence of 6 mM extracellular [Ca2+], stimulation with ACh [79] or Ins(1,4,5)Pa [8] causes both intracellular Ca2+ release (as discussed above) and a prolonged influx of extracellmar Ca2’ (10-30 mm). This $flux can be re;:rsibly blocked by remo$ng Ca2+0 or adding Ca channel blockers @$I+ or Co ) to the extracellular bath 17, 8, 791. 0 influx increases in oocytes where the intracellular stores have been de leted [79]. This !t suggests that the amount of Ca present within intracellular stores regulates Ca2+,, entry. Extensive junctions between the ER and the cytoplasmic membrane [58] could allow Ca2’, to enter the ER directly. Thus, by controlling the amount of stored Ca2’, InsP3 could indirectly influence Ca2’, entry [801.

220

CELL CALCIUM

A lns(2.4,5)P3

0

200

400

600

B

Fig. 1 Cb-actetitics

800

1000

1200

1400

1600

Time (s)

of Ins(2,4,5)PGnduced

[Ca’+]i oscillations in Xenopus oocytes.

intracellular injection of 0.5 pmole of Ins(2,4,5)fi.

(A) Cl- cumzntoscillations

in response to an

The more negative the current, the higher the intracellular Ca2’ concentration

The

insert is an enlarged segment of the tmcing (taken from 380-450 s) showing high-frequency, low-amplitude oscillations superimposed on the low-frequency, high amplitude oscillations.

(B)

Three-dimensional representation of the frequency (Y-axis) and amplitude

(Black - lowest, White - highest) domains of the tracing shown in comprises

several different oscillating

frequencies.

A as

a function of time (X-axis).

At any given time, the signal

As time passes, frequency decreases while amplitude increases.

The high

frequency, low amplitude oscillations (shown in A) gradually disappear over time

Inositol (1,3,4,5)-tetrakisphosphate (InsP4), a direct metabolite of Ins(1,4,5)P3, may regulate Ca2+, iuflux in some cells (for a review, see [81]). In the oocyte, microinjection of Ins(2,4,5)P3 (a synthetic InsP3 isomer not expected to be metabolized to InsP4 [82]) produces Ca2’, influx while IusP4 does not [8]. Although this suggests that InsP4 is not required for Ca2+, entry, the proposed synergy between Ius(1,4,5)P3 and InsP4 has not been ruled out.

[d”]i

oscillations

[Ca2+]i oscillations have now been described in several different cells in response to a wide variety of stimuli and their frequency may encode a signal

for the regulation of cellular fimction [83]. In Xenopzu oocytes, Cl- current oscillations were found when the endogenous ACh receptors were originally described [23]. Since then [Ca2+]I oscillations that do not depend on ca2’, ehtIy have been described in response to fertilization [84], stimulation of mRNA-expressed receptors [39], injections of Ius(1,4,5)P3 [6-g, 10, 15, 23, 24, 47, 601, InsP4 [7, 8, 853, Ins(2,4,5)P3 [7] and inositol 1,4,5-trisphosphothioate (Ins(l,4,5)PS3) [7, 86, 871. Oscillationscharacteristics: [Ca2+]ioscillations only occur within an InsP3 concentration window, i.e. too much or too little InsP3 do not produce oscillations [7]. Within that ‘window’, the frequency (but not the amplitude) at which [Ca2+]i

XEHOPUS OCICYES

: THE 4 DIMENSIONS OF Ca SIGNALLING

oscillates is directly proportional to the amount of injected hSP3 171. This observation may explain why increasing concentrations of cell-surface agonists cause [Ca2+]i oscillations of increasing frequency: cell-surface agonists increase InsP3 levels in a dose-dependent manner, and InsP3 increases the frequency at which [Ca2+]l oscillates, also in a dose-dependent manner. High doses of InsP3 give rise to sinusoidal [Ca2+]i oscillations whereas low doses of Ins(1,4,5)P3 often produce transient [Ca2+]i elevations arising from a stable baseline [71. Both types of oscillations can be observed within the same response (Fig. 1A). Thus, and contrary to what has been proposed 1881, [Ca2+]i oscillations of various shapes may arise from a common mechanism. Detailed analysis reveals complex oscillatory patterns. Figure 1A discloses the Clcurrent response to a microinjection of Ins(1,4,5)P3. Intuitively, it appears that oscillation frequency diminishes over time while amplitude increases. This is confirmed by power spectral analysis (Fig. 1B). The magnified section in Figure 1A illustrates that several low amplitude oscillations lie within each large amplitude oscillation. These low amplitude, high frequency oscillations can be seen early in the response and disappear over time, as shown in Figme 1B. The gradual synchronization of independently releasing Ca2+ stores might explain why the small oscillations disappear while the amplitude of the large oscillations increases. Proof of this hypothesis, however, will require knowledge of the spatial distribution of intracellular Ca2+. Mechanism of [Cd+]i oscillations : Ins(2,4,5)P3 and Ins(l,4,5)P3S3 cause [Ca2+]i oscillations while their levels remain constant at least 15 min after injection [7]. This observation, along with the dose-frequency relationship described above, suggest that [Ca2’]i oscillations are not due to oscillations in Ins(1,4,5)P3 levels but result from a mechanism distal to the Ins 1,4,5)P3 generation site. 1 Either large doses of Ca ’ [5, 891 or repeated injections of small doses of Ca2’ [ 151 can cause However more modest Ca2+ Ca2+ oscillations. injections (to cause Cl--curLnts similar to those induced by InsP3) do not elicit oscillations [71. Hence, Ca2+-induced Ca2+ release may not be sufficient to cause [Ca2+]i oscillations; involvement of the InsP3-sensitive calcium pool is probably

221

required. Intracellular injection of Ca2’ decreases the amplitude without affecting the frequency of on-going InsP3-induced [Ca2’]i oscillations VI. This is in keeping with the feedback inhibition by Ca2’ on InsP3-induced Ca2’i release [ll]. Depletion of the Ca2’ stores, which mainly affects oscillations frequency in other cells [go], has not been studied yet Ca2’ waves Ca2’ is often released from a discrete intracellular locus [21, 26, 55, 58, 91-941 and may then distribute itself to the rest of the cell in the form of a wave [95-971. This phenomenon, which had previously been seen in muscle and neuronal cells, has recently been described in several ‘non-excitable’ cells (see ref. in [98]). As with medaka [99] and frog [NO] eggs, fertilization of Xeno us eggs causes a subcortical Ca2+ wavefront 2P (Ca activity around 1.2 @VI)which travels away from the site of sperm entry at a velocity of about 10 pm/s [62, 1011. Stimulation of endogenous and mRNA-expressed receptors linked to the InsP pathway can also result in a propagated Ca2+ wave ml. Although suspected, the contribution of InsP3-insensitive stores to the mechanism of these Ca2’ waves has not yet been studied in Xenopus.

Ca2’ signaling in Xenopus oocyte: A model Clearly, much remains to be learned about the molecular mechanisms responsible for [Ca2’]i oscillations and Ca2’ waves. The following model attempts to link the known characteristics of the InsP3-sensitive Ca2’ stores and IM%induced Ca2’ release (summarized in Fit 2) to the behaviour of [Ca2’]i oscillations and Ca ’ waves. This model is meant to help formulate hypotheses. Quite likely, it represents a gross oversimplification (the fust three phases are schematized in Fig. 3). Phase I: Latency

Following membrane receptor stimulation, activated G-proteins trigger the inositol phosphate second messenger cascade. Ins( 1,4,5)P3 level increases

CELL CALCIUM

222

Phase 3: Calcium reuptakepredominance

ACh

The rise in [Ca2+]i inhibits further InsP3-induced and the Ca2t-ATPase and Ca2+ release, Ca2+-extrusion mechanisms lower [Ca2’]i. At a given [Ca2’]i, the InsP3 level which released Ca2’ during Phase 2 (point ‘A’, Fig. 3) is now unable to do so (point ‘B’, Fig. 3). This hysteresis, which is required if the system is to oscillate, may result from the slow reversibility of the Ca2+ inhibition [681. With the low [Ca2+]i and the continual presence of Ins(1,4,5)P3, the system becomes disinhibited and cycles back to Phase 2. Note that within an oscillatory cycle, the rate of Ca2+ removal closely matches the rate at which Ca2+i was released (see Fig. 1A). Since the rate of Ca2’ release is proportional to [InsPs], a higher [InsPs] will yield a higher frequency of oscillation, provided that the feedback inhibition by Ca2’ remains operative. Phase 4 : Synchronization

Fig. 2 Summary of inositol phosphate&?+ oocytes (see text for details). (AC%), and membrane Ins(l,4,5)P3

(hrsPs).

(membrane-bound extracellular increases

receptor Imp3

store = rounded

the free intracellular release

Cl- channels. before

signaling

Ca”

in Xerwpus

in this case acetylcholine

interaction

releases

ultimately

stored facilitated

Ca2+ also inhibits

*Fh

Ca2+

influx of

by hrsP4).

concentration

A

produces

intracellular

box) and stimulates

Ca2’ (the latter perhaps

the Ca2+-gated induced

Agonist,

When integrated by the Cl- current assay, the initial chaotic release of Ca2+ from several independent

This

and stimulates its own InsP3-

it is taken back into the stores via an

ATPase

graduahy towards the threshold necessary to release stored Ca2+. Phase 2: Calcium release predominance With three or more Ins(1,4,5)P3 molecules bound to each InsP3-receptor [60], Ca2+ contained in one or several discrete stores is suddenly released. The rate of this release momentarily surpasses the combined capacity of the Ca2’-ATPase and Ca2+-extrusion mechanisms, and [Ca2+]i rises. A high [Ca2+]i wavefront then propagates across the cell, perhaps by inducing release of Ca2’ from subcortical InsP3-insensitive stores.

Time Fig. 3 Schematized (X-axis) following arrow). [Ca2+]i.

A more negative Phase

of Cl- current (Y-axis) over time

1

=

c1- current Latency,

with acetylcholine represents

Phase

Phase

3 = Ca2+ reuptake

mechanisms

that might account

predominance, molecular

oscillations

cellular stimulation

presented in the text

2

-

(ACh,

an increase

in

Ca2+

release

predominance.

The

for these phases

are

XENOPUS OOCYTES : THE 4 DIMENSIONS

223

OF Ca SIGNALLING

stores result in a high-frequency, low-amplitude signal. The combined inhibit0 effect of local Ca2+ ?+ diffusion and propagated Ca waves gradually Their synchronize the InsP3-sensitive stores. simultaneous Ca2+ release results in a signal of higher amplitude. Phase 5 : Termination The signal may terminate via two different mechanisms: (a) metabolic degradation of InsP3 (this is probably the case when small amounts of InsP3 are injected); (b) desensitization (this happens with high doses of InsP3, and arises at a site distal to InsP3 production but proximal to the Ca2+-gated Cl- channels).

Future directions The Xerwpus oocyte has already proven invaluable in dissecting the inositol phosphate pathway. Perhaps because of the archaic nature of this signaling system, there is little doubt that the observation made in the oocyte will continue to have widespread biological significance. Its large size, which is otherwise so attractive, entails a spatially complex Ca2+ si nal. Hence, high 2% resolution and high speed Ca imaging is likely to reveal a host of previously unsuspected information. Cloning of the enzymes responsible for the metabolism of Ins(1,4,5)P3 [119, 1201 and judicious use of site-directed mutagenesis will provide powerful tools to further define this enormously complicated pathway.

Physiological function Oocytes mature into eggs that are then fertilized to become embryos The role of Ca2’ in the complex oocyte maturation is controversial (for [2, 17, 102, 1031, against [69, 104-1061). However, recent data implicate Ca2+ in the signal transduction of a maturation promoting factor [22, 1071. The egg polarity may be maintained through a continuous Ca2to-dependent Cl- current, flowing from the animal to the vegetal pole [2, 1081. Egg fertilization potential can be mimicked by Ins(1,4,5)P3 [26, 641, and Ca2+ waves may play a role in cortical granule exocytosis [62, 109], ooplasmic segregation [92] and protection against polyspermia [llO]. Lithium (Lit) inhibits the recycling of Ins(1,4,5)P3 [98] and has teratogenic effects in the developing Xenopus embryo [ill]. Its microinjection into prospective ventral cells results in supernumerary dorso-anterior structures such as the neural tube and the eyes. These ventral cells can be rescued when myo-inositol is coinjected with Lit [112]. Although the mechanisms involved are far from being elucidated - InsP3 may be involved in the signal transduction of 2powth factor-like molecules [113-1181, and Ca is involved in mitotic cleavage (see [22] for references) these important experiments suggest that the inositol phosphate pathway has far reaching roles in growth and development.

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