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Journal of Physiology (1992), 458, pp. 307-318 With 6 figures Printed in Great Britain
MECHANISM OF RELEASE OF Ca2+ FROM INTRACELLULAR STORES IN RESPONSE TO IONOMYCIN IN OOCYTES OF THE FROG XENOPUS LAEVIS
BY SHIGERU YOSHIDA* AND SUSAN PLANTt From the Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, the Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan and the Department of Preclinical Veterinary Sciences, University of Edinburgh, Summerhall, Edinburgh EH9 1QH (Received 28 August 1991) SUMMARY
1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole-cell currents using the twoelectrode voltage-clamp method. 2. The extracellular application of ionomycin, a selective Ca2+ ionophore, evoked an inward current consisting of a spike-like fast component followed by a long-lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose-dependent manner and was dependent on Cl-. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0 1 ftM). In contrast, a larger ionomycin response (1 ,lM), consisting of fast and slow components, was followed by refractory period. Washing for 50-90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA AM, 1-10 ,tM), a membrane-permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX, 0-3-2-0,tg/ml), a blocker of guanine nucleotide-binding regulatory proteins (G proteins). In contrast, the response to acetylcholine (ACh), which is known to occur via a G protein, was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mm EGTA), whereas the slow component was suppressed. 7. Injection of inositol 1,4,5-trisphosphate (IP3) following a response to extra* Present address: Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan. t Present address: Department of Physiological Sciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH.
MS 9672
S. YOSHIDA AND S. PLANT cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast, ionomycin evoked a small inward current when it was applied after an inward-current response evoked by 1P3 injection, whereas a second injection of 1P3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins, resulting in activation of Ca2+-activated Clchannels, (b) ionomycin mainly acts on the same intracellular Ca2+ stores as 'P3, and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response, whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores. 308
INTRODUCTION
Xenopus oocytes have been widely used for functionally expressing receptors and ion channels by injecting exogenous mRNAs isolated from a variety of preparations to determine their amino acid sequences and also for investigating endogenous receptors for acetylcholine (ACh) etc. (see Dascal, 1987; Snutch, 1988 for reviews). In these cases, receptors are coupled to endogenous pathways in Xenopus oocytes, which elicit inward-current responses consisting of two components, a rapid transient and a long-lasting slow component. All these receptor mediated current responses have been found to operate via Ca2+-activated Cl- channels. Stch Clchannels are activated when the concentration of intracellular Ca2+ is increased, mainly by the release of Ca2+ from intracellular stores. Xenopus oocytes have been used as a convenient material for studying the mechanism of Ca2' release. Recently, the quanta4 and spacio-temporal aspects of Ca2+ release in Xenopus oocytes have been studied in highly localized regions using laser confocal microscopy. Local Ca2+ release occurred in an all-or-none mariner in response to photoreleased inositol 1,4,5-trisphosphate (1P3), suggesting that Ca2+ release from larger areas is a collection of quantized local Ca2+ release (Parker & Ivorra, 1990). Examining muscarinic ACh receptors, which are coupled to turnover of phosphatidyl inositol, Lechleiter and his co-workers (Lechleiter, Girard, Peralta & Clapham, 1991b) found regenerative spiral waves of retease of free Ca2+, Further study revealed differences in timing and magnitude of Ca2+ release among subtypes of muscarinic ACh receptors (Lechleiter, Girard, Clapham & Peralta, 1991 a). Although, these studies clarify some aspects of intracellular release of Ca2+, the mechanism of increase in the intracellular concentration of Ca2+ ([Ca2+]i) in response to ligands is still a matter of debate. The action of A23187, a Ca2+ ionophore, has been investigated in Xenopus oocytes (Gillo, Lass, Nadler & Oron, 1987); pretreatment of oocytes with A23187 resulted in depletion of intracellular Ca2+ stores. It has also been reported that A23187 elicited a slow inward current in Xenopus oocytes (Boton, Dascal, Gillo & Lass, 1989). However, A23187 was mainly used to permealize oocytes to Ca2+, and no work was attempted to study the action of A23187 itself on Ca2+. The aim of the present work is to study the mechanism of elevation of [Ca2+]i using another Caa2+ ionophore, ionomycin. Ionomycin is known to release Ca2+ from intracellular stores in various types of cells (Liu & Harmann, 1978; Boynton, Cooney, Hill, Nilsson, Arkhammar & Berggren, 1989). It was found that ionomycin elicited an inward current response,
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consisting of fast and slow components, and mimicked receptor-mediated responses. The ionomycin response was also similar to current responses evoked by the injection of 1P3 (Oron, Dascal, Nadler & Lupu, 1985; Gillo et al. 1987). METHODS
Preparation of Xenopus oocytes. Mature oocytes, at stages V and VI (Dumont, 1972), were collected from the ovary of adult female frogs of Xenopus laevis which were anaesthetized by cooling with ice (Yoshida & Plant, 1991). Oocytes were freed from the connective tissue and follicle cell layer with 2 mg/ml collagenase (Sigma, Type IA) in Ca2+-free saline for 1-5-2-5 h at room temperature (20-23 °C). These 'defolliculated' or 'denuded' oocytes were stored at 19 'C in modified Barth's solution supplemented with penicillin (100 IU/ml), streptomycin (100 lOg/ml), 0-25 -nm sodium pyruvate and 2 % Ficoll (Sigma) before electrophysiological study. Electrophysiology. The conventional two-electrode voltage-clamp method, using a home-made voltage-clamp system, was used to measure whole-cell currents in Xenopus oocytes as described previously (Yoshida, Plant, Taylor & Eidne, 1989; Yoshida & Plant, 1991). DC resistances of membrane potential-recording and current-injection electrodes, filled with 3 M KCl, were 1-3 MQ and 08-1-5 MQ, respectively. Membrane currents were processed by a digital audioprocessor (SONY PCM-701ES; sampling frequency 44-1 kHz) modified to accept down to DC and stored on videotapes with a video cassette recorder (Panasonic NV-G12B). The stored data were displayed on a chart recorder (Graphtee SR6335-2L) for illustrations. The reversal potential for current responses to ionomycin was estimated by the ramp method (Yoshida & Plant, 1991); repetitive voltage ramps, 0-8-1 0 s in duration with slopes of 80-120 mV/s, were applied to an oocyte during a response to ionomycin. Solutions. The ionic composition of the standard solution used for electrophysiology was: 115 mM NaCl, 2 mm KCl, 1-8 mm CaCl2, 1 mM MgCl2 and 10 mm HEPES (pH 7 4). Chloride- was replaced with the membrane-impermeant anion methanesulphonate- (Aldrich), and Na+ was replaced with Tris+ (Sigma). lonomycin (free acid form), a Ca2+ ionophore, was purchased from Calbiochem (LaJolla, CA, USA). The acetoxymethyl ester of bis-(O-aminophenoxy)-ethaneN,N,N',V'-tetraacetic acid (BAPTA AM; Calbiochem), a membrane-permeable intracellular Ca2+ chelator, was used to chelate intracellular Ca2+ (Barritt & Lee, 1985). lonomycin and BAPTA AM were firstly dissolved in dimethyl sulphoxide (DMSO; Sigma); the final concentration of DMSO in the bathing medium was between 0 1 and 0 5 % (v/v). DMSO had no effect on its own at these concentrations. Ethylene glycol bis-(/?-aminoethylether)-N,N,N'N'-tetraacetic acid (EGTA; a Ca21 chelator), inositol 1,4,5-trisphosphate (JP3, potassium salt) was obtained from Sigma. Injection of chemicals into oocytes was carried out using a pressure-injection system (Hamilton Microlab-M, Switzerland). Pertussis toxin (PTX) was obtained from Calbiochem and Sigma. When necessary oocytes were treated with PTX (0-3-2-0 ,ug/ml) for 16-19 h in the standard solution which was supplemented with a stabilizer, bovine blood albumin (0 3 mg/rnl; Wako Pure Chemical Industries, Japan) (Cohen-Armon, Sokolovsky & Dascal, 1989). The pH of all solutions was adjusted to 7 4, and solutions were introduced to and removed from the experimental chamber by perfusion. Statistical analysis. Since responses to chemicals varied from frog to frog (Yoshida & Plant, 1991), oocytes from a single donor were used in the present study to examine the effects of ligands. Also, as the number of observations in the present work was smaller than 30, the t test, one of the parametric tests, was not suitable because it required normal distribution and homogeneity of variance. Therefore, the Mann-Whitney U test (two-tailed with a significance limit of 0-05), one of the non-parametric tests (or distribution-free statistics), was used for the statistical analysis of two independent groups (Phillips, 1978). The Mann-Whitney U test needs only nominal or ordinal scale data and does not require normal distribution or homogeneity of variance. RESULTS
Current responses to ionomycin The resting potential of defolliculated Xenopus oocytes measured with a single voltage-recording electrode in standard solution ranged between -47 and -76 mV, and the average value from representative oocytes was - 618 +8-2 mV (mean +
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A 01 ,uM IM
C
B
5uM IM
1,uM IM
D 05,uM ACh
I100 nA
1000QnA (C)
5 min
Fig. 1. Inward current responses to ionomycin (IM; A-C) and acetylcholine (ACh; D) in defolliculated oocytes of Xenopus laevis. The two-electrode voltage-clamp method was used to record whole-cell currents from the oocyte. The holding potential was -60 mV in all cases. The application of IM and ACh is indicated by horizontal bars. Solutions were introduced to and drained from the experimental chamber by perfusion. The perfusion dead time was less than 2 s in all experiments. Downward deflections indicate inward currents in all figures. The amplitude calibration of 100 nA is for A, B and D, and 1000 nA for C. The time scale applies to all records. A 1 aM ionomycin 30nmn
r60m
B 01 ,uM ionomycin 100 nA 40 nA
(A) (B)
30 min
Fig. 2. Reproducibility of the ionomycin response. A, 1 ,4M ionomycin was repetitively applied to the same oocyte. The application of ionomycin is indicated by horizontal bars. Parallel bars show interval of the record. B, 0 1 4UM ionomycin was repetitively applied to the oocyte as indicated by horizontal bars. Note that the current calibration of 100 nA is for A, and 40 nA for B.
S.E.M.; n = 20). Therefore, oocytes were routinely voltage clamped at -60 mV unless otherwise stated. In defolliculated Xenopus oocytes, ionomycin evokes an inward-current response which is dependent on the release of Ca21 from intracellular stores. A typical example is shown in Fig. 1 A-C. The extracellular application of ionomycin (IM) by perfusion (indicated by horizontal bars) evoked inward-current responses in a dose-dependent manner. With a low concentration of ionomycin the response was a slow inward current (Fig. IA). The response consisted of a spike-like fast component followed by
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a smooth slow component when the concentration of ionomycin was increased (Fig. 1 B and C). When the ionomycin concentration was below 1 ,CM, the amplitude of the ionomycin response did not exceed 1000 nA. At higher concentrations of ionomycin, e.g. 5jM, the response could be larger than 1000 nA as shown in Fig. 1 C. The mean values of the peak amplitude of the ionomycin response (the peak amplitude of the fast component when it is obvious) were approximately 30 nA (0 1 /M ionomycin; n = 3), 80 nA (0 5 /SM ionomycin; n = 5), 330 nA (1 /LM; n = 5), and 3010 nA (5 sM; n = 5). The estimated EC50 was approximately 1-7 #M. The ionomycin responses were similar in shape to current responses which are evoked by the activation of receptors like the endogenous ACh receptor for example (Fig. 1D); the application of ACh (horizontal bar) produced an inward-current response which also consisted of two components. The differences are (a) that the ACh response showed current oscillations superimposed on the slow component (this feature is prominent in the oocyte shown in Fig. 4B) and (b) that washing-out of ionomycin from the extracellular perfusate could not abort the ionomycin response (Figs lB and 4A), whereas the ACh response subsided when ACh was removed from the bathing solution (Figs ID and 4B and C). A refractory period followed the ionomycin response when it was large in size and consisted of fast and slow components (n = 7). A representative example is shown in Fig. 2A; the records were taken from the same oocyte. A response was elicited by the first application of 1 ,UM ionomycin. The second application of ionomycin during the slow component failed to evoke any additional current. Jonomycin was washed out from the bathing medium and the oocyte was challenged by ionomycin for a third time, resulting in an incomplete recovery, but the response consisted of fast and slow components. A full recovery of the ionomycin response was obtained by a prolonged wash. Usually, such a full recovery was observed when oocytes were washed for 50-90 min. In contrast, no appreciable refractory period was observed when lower concentrations of ionomycin were used to elicit inward currents. An example is shown in Fig. 2B. Repetitive application of 01 /SM ionomycin with a short interval evoked inward-current responses similar in size and shape. Such responses were small in size and did not show distinct fast and slow components. Ionic dependence of the ionomycin response In order to examine which ions were responsible for responses to ionomycin, the reversal potential for the ionomycin-evoked current was measured using the ramp method (see Methods). The measured value was around -25 mV (- 25-7 + 3-3 mV; n = 3) and was compatible with the reversal potential for Cl- in Xenopus oocytes (Dascal, 1987; Yoshida et al. 1989). Furthermore, a depolarizing shift of approximately 17 mV was observed in the reversal potential of the ionomycin response when the extracellular Cl- concentration was halved (Yoshida et al. 1989). This shift is in good agreement with the expected value of 17-5 mV given by the Nernst equation. The results indicate that the current response evoked by ionomycin is predominantly dependent on Cl-. 11
PHY 458
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Effect of an intracellular Ca2+ chelator BAPTA on the ionomycin response It is established that the Cl- channels, which are responsible for current responses to ACh, serotonin, substance K etc. in Xenopus oocytes, are activated when the concentration of intracellular Ca2+ is increased, i.e. Ca2+-activated Cl- channels A Control
B BAPTA AM
1100 nA 5 min Fig. 3. Effect of extracellularly applied BAPTA AM, a membrane-permeant intracellular Ca2+ chelator, on the current response to ionomycin. The applications of ionomycin are indicated by horizontal bars. A, a control response to 1 /,M ionomycin. B, a response to 1 /M ionomycin in an oocyte which was pretreated with 10/tM BAPTA AM for 2 h. The time and the current calibrations apply to both responses.
(Dascal, 1987; Snutch, 1988). As the response to ionomycin is similar to receptormediated responses and is dependent on Cl-, the same channel would appear to be involved. This possibility was examined by chelating intracellular Ca2` using bis-(Oaminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a pH-independent chelator specific for Ca2+. The acetoxymethyl ester of BAPTA (BAPTA AM) was used in the present study because it is membrane permeant, and when it penetrates the cell membrane, it is cleaved by cytoplasmic esterases to yield free BAPTA and chelate intracellular free Ca2+; de-esterified BAPTA is membrane impermeant and locked within the cell (Tsien, 1981). Since no significant differences were observed in the chelating effect of intracellular free Ca2+ by extracellularly applied BAPTA AM in the presence or absence of Ca2+ in the bathing medium (Barritt & Lee, 1985), oocytes were treated with BAPTA AM in standard solution. Ionomycin (1 /tM) was applied to oocytes which had been preincubated with 1 or 1O /LM BAPTA AM for 1-3 h. At 1 ,UM, BAPTA AM did not show significant suppression of the ionomycin response (ni = 5; not illustrated). In contrast, when the concentration of BAPTA AM was increased to 10 ,sM, the ionomycin response was markedly reduced (n = 3). The mean values of the peak amplitude of the fast and slow components of the response to 1 ,IM ionomycin in control oocytes not treated with BAPTA AM (see Fig. 3A for an example) were approximately 110 and 30 nA, respectively (n = 3). Figure 3B displays a response to 1 ,UM ionomycin recorded from an oocyte pretreated with 10 /SM BAPTA AM for 2 h; ionomycin evoked a small and slow inward current without appreciable fast current in this case. The mean peak amplitude of the ionomycin response in such BAPTA AM-treated oocytes was 12 nA (n = 3). The oocytes illustrated in A and B were obtained from the same donor frog. Since BAPTA is a Ca2+ chelator and reported to prevent the transient increase in intracellular free Ca2+ (Barritt & Lee, 1985), it is concluded that the ionomycin response is dependent on the rise in the intracellular free Ca2+ concentration.
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Effect of a blocker of G proteins, pertussis toxin (PTX), on the ionomycin response It has been reported that receptor-mediated responses in Xenopus oocytes act via guanine nucleotide-binding regulatory proteins (G proteins) which are sensitive to pertussis toxin (PTX), e.g. acetylcholine and serotonin receptors (Dascal, Ifune, A PTX (2,g/ml) B Control IM ACh
C PTX (03,ug/ml) Ach
1100 nA 5 min
Fig. 4. Effect of pertussis toxin (PTX) on ionomycin (IM)- and acetylcholine (ACh)evoked responses. A, a response to 1 ,UM ionomycin in an oocyte which was pretreated with PTX (2 ,ug/ml, for 18 h). B, a control response to 05 /M ACh. C, a response to 05 #M ACh recorded in an PTX-pretreated oocyte (03 ,ug/ml, for 18 h).
Hopkins, Snutch, Liibbert, Davidson, Simon & Lester, 1986; Nomura, Kaneko, Kato, Yamagishi & Sugiyama, 1987; Cohen-Armon et al. 1989). These receptormediated responses are dependent on the opening of Ca2+-activated Cl- channels. Although pertussis toxin is reported to inhibit receptor-mediated responses, it did not exert any appreciable effect on the ionomycin response; inward-current responses similar in size and shape were recorded from oocytes which were pretreated with 03 ,tg/ml PTX (n = 4; not illustrated) and even with a high concentration (2 /Lg/ml) of PTX (n = 4). The oocyte shown in Fig. 4A was pretreated with 2 ,sg/ml PTX for 18 h. In contrast, pretreatment of oocytes with 03 ,ug/ml PTX was sufficient to suppress the ACh response (Fig. 4C; n = 4) and no appreciable current was observed at 2 gg/ml PTX (n = 3). The results indicate that G proteins are not involved in the ionomycin response. The dependence of the response to ionomycin on extracellular Ca2+ The ionomycin response could be also elicited in Ca2+-free bathing medium as shown in Fig. 5. Extracellular Ca2+ was removed from the bathing medium and any residual Ca2+ was chelated by 2 mm EGTA. Since the oocyte membrane became too leaky to be voltage clamped in this EGTA-containing Ca2+-free solution, divalent cations were used to replace Ca2+. A control response in standard solution (1-8 mm Ca2+) is displayed in Fig. 5A. As shown in Fig. 5B (Mn2+ substituted for Ca2+) and C (Ba2+ substituted for Ca2+), the amplitude of the fast component of the ionomycin-evoked response did not significantly change, although the fast component frequently showed more prominent multiple peaks, in the absence of extracellular Ca2+. The application of 1 /UM ionomycin is indicated by a horizontal bar in each current trace. In contrast, the slow component was significantly reduced in size under Ca2+-free conditions. The aveyage peak amplitudes of the fast and slow components of responses evoked by 1 ,UM ionomycin were 212 and 152 nA (n = 5) in standard 11-2
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S. YOSHIDA AND S. PLANT
solution, respectively, whereas these values were 228 and 17 nA (Mn2+; n = 4), and 204 and 17 nA (Ba2+; n = 4), respectively. There was no significant change in the amplitude of the fast component of the response. On the contrary, the slow component reduction was statistically significant (P = 0-05) when Ca21 was removed A Control
8 Mn2+
C Ba2+
1200nA 5 min
Fig. 5. Current responses evoked by ionomycin under Ca2+-free conditions. A, a control response evoked by 1 /CM ionomycin in standard solution. B, a current response to 1 /,M ionomycin in Ca2+-free solution; the Ca2+ of the standard solution was replaced with an equimolar amount of Mn2+. C, an inward-current response elicited by 1 ,UM ionomycin in bathing medium where Ca2+ was replaced by Ba2+. Both solutions used for B and C contained 2 mm EGTA to chelate residual Ca2+. Horizontal bars indicate the applications of 1 I/M ionomycin.
from the bathing medium. These results indicate that the fast component is not dependent on extracellular Ca2 , whereas the slow component appears to be somewhat dependent on the entry of Ca2+ from the extracellular solution. Interaction between ionomycin and IP3 in inducing Ca2+ release from the stores, The results obtained using an intracellular Ca2+ chelator BAPTA indicate that a current response evoked by ionomycin is dependent on an increase in the concentration of intracellular free Ca21 (see Fig. 3). It has been reported in various types of preparations, including Xenopus oocytes (Gillo et al. 1987; Berridge, 1988), that an intracellular second messenger inositol 1,4,5-trisphosphate (IP3) acts on the intracellular Ca2+ stores, resulting in the release of Ca2+ (for review see Berridge, 1987; Berridge & Irvine, 1989; Rana & Hokin, 1990). Figure 6A shows that the application of ionomycin (5 /tM) evoked an inward current, with a short latency (horizontal bar marked IM), consisting of fast and slow components. A third glass micropipette filled with 10 /tM IP3 solution was inserted into the oocyte during the slow component of the ionomycin response (downward arrowhead). The pipette insertion caused some damage to the oocyte which was reflected by a jump in the holding current. Then, a single pulse of 50 nl of 10 /tM IP3 solution was delivered by the pressure-injection system into the oocyte (horizontal bar marked IP3) following the ionomycin response, but no significant change was observed in the membrane current (n = 4). This failure in evoking a response by injected IP3 is not due to damage caused by the insertion of the third micropipette because oocytes which had not been previously challenged by ionomycin produced an inward-current response upon injection of IP3 delivered through the third electrode. It is suggested that ionomycin acts on the same intracellular Ca2+ store as IP3 and that the response to ionomycin depletes this store of Ca2+.
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In Fig. 6B, a third electrode containing 10 JM IP3 was inserted into an oocyte and the oocyte membrane was allowed to seal for a few minutes. Then 50 nl of 10 #M IP3 solution was injected by pressure into the oocyte (horizontal bar marked IP3), resulting in an inward current consisting of fast transient and slow components. A IM
B IP3 I
IM
1100 nA 5 min
Fig. 6. Interaction of ionomycin (IM) and inositol 1,4,5-trisphosphate (1P3) in inducing current responses. A, an oocyte was challenged with 5 /M ionomycin and produced an inward current consisting of fast and slow components. A third electrode containing IP3 was inserted into the oocyte during the slow phase of the ionomycin response (downward arrowhead) and 50 nl of 10 #M IP3 was injected. B, an oocyte was firstly challenged with IP3 which was delivered through a third electrode followed by the extracellular application of 5 uim ionomycin. The records A and B were obtained from different oocytes.
When ionomycin (5 ,tM) was applied in succession (horizontal bar marked IM), a small inward current was evoked (n = 3). On the contrary, no substantial current was evoked when oocytes were challenged by a second injection of IP3. DISCUSSION
The present work has shown that ionomycin (free acid form), a selective Ca2+ ionophore, evoked an inward-current response consisting of a spike-like fast current followed by a long-lasting slow current. This ionomycin response mimicked receptormediated responses observed in Xenopus oocytes (Fig. 1). The ionomycin response was also similar to the response elicited by the injection of inositol 1 ,4,5-trisphosphate (IP3; Fig. 6B) (Oron et al. 1985; Gillo et al. 1987). Furthermore, the ionomycin response appeared to share the same mechanism as receptor-mediated and IP3induced responses, i.e. ionomycin activated Ca2+-activated Cl- channels via release of Ca2+ from intracellular stores. There are, however, several differences between the ionomycin and receptor-mediated responses. (a) The involvement of IP3 may not be important in the ionomycin response because the ionomycin response showed very few current oscillations; it has been reported that receptor-mediated responses which occur via the phosphoinositide pathway and responses evoked by the injection Qf 1P3 show current oscillations (fluctuations) superimposed on their slow component (e.g. Dascal, Gillo & Lass, 1985; Gillo et al. 1987). (b) G proteins do not seem to be involved in the ionomycin response (Fig. 4). Blood sera from vertebrate species are reported to elicit oscillatory Cl- currents in Xenopus oocytes (Tigyi, Dyer, Matute &
S. YOSHIDA AND S. PLANT Miledi, 1990). In the present study, 0 3 mg/ml bovine blood albumin (Fraction V; Wako Pure Chemical Industries, Japan) was used as a stabilizer (see Dascal et al. 1986; Cohen-Armon et al. 1989) for testing the effect of pertussis toxin (PTX). The possibility that the inhibition of the ACh response was not due to the blockade of G proteins by PTX but by depletion of the intracellular Ca2" stores by albumin can be excluded because (a) the albumin used in the present study did not elicit any currents on its own (n = 5), (b) ionomycin induced inward-current responses in Xenopus oocytes which were treated with albumin and PTX (Fig. 4), and (c) the ACh response was also inhibited by PTX in oocytes not treated with albumin (n = 3) (see Nomura et al. 1987). 316
Mechanisms underlying the fast and the slow components of the ionomycin response Differences were revealed in the properties of the fast and the slow components of the ionomycin response. The fast component was not substarwtially affected by removing Ca2+ from the bathing solution or by replacing extracellular Ca2+ with Ba2+ or Mn2+, whereas the slow component was significantly suppressed (Fig. 5). The data suggest that entry of Ca2+ from outside the oocyte, including Ca2+ from the plasma membrane (see below), considerably contributes to the slow but not to the fast component of the ionomycin response and that the fast component is predominantly dependent on an intracellular Ca2+-releasing mechanism. Moreover, the fast component of the ionomycin response frequently showed multiple peaks (Figs 1B and 5A), and this was potentiated in the absence of external Ca2+ (Fig. 5), whereas the slow component did not show distinct peaks. The multiple peaks of the fast component suggest (a) ionomycin acts on distinct types of Ca2+ stores with time, e.g. superficially and deeply located Ca2+ stores or (b) Ca2+ which is released by ionomycin causes a cascade of Ca2+-induced Ca2' release. When the intracellular Ca2+ was chelated by BAPTA, an intracellular Ca21 chelator, the fast component of the ionomycin response was greatly suppressed leaving a slow current (Fig. 3). It is reported that higher doses of EGTA, another Ca2+ chelator, are necessary to inhibit the slow component of ACh and serotonin responses than to suppress the fast component inXenopus oocytes (Boton et al. 1989). It was proposed by Gillo and his co-workers (1987) that (a) the fast component of such responses represents rapid interaction of Ca2+ with Cl- channels and requires a relatively high Ca2+ concentration, and (b) the slow component proceeds through the activation of some biochemical event which requires a lower Ca2+ concentration. Also, they speculate that the two components may be mediated by distinct Clchannels (Gillo et al. 1987; Boton et al. 1989).
Actions of ionomycin and IP3 on the intracellular Caa2+ stores It has been reported that injection of inositol 1,4,5-trisphosphate (IP3) mimics receptor-mediated current responses in Xenopus oocytes (Oron et al. 1985; Gillo et al. 1987). The present work shows that the response evoked by ionomycin is similar to both receptor-mediated and lP3-elicited responses. The results displayed in Fig. 6 indicate that ionomycin shares a common intracellular Ca2+ release mechanism with IP3, as no substantial current was elicited by IP3 following the ionomycin response (Fig. 6A). It is unlikely that this effect was due to desensitization of Ca2+-gated Cl-
MECHANISM OF Ca2+ RELEASE FROM STORES 317 channels upon the elevation of intracellular free Ca2+ concentration by ionomycin, because repetitive injections of Ca2+ have been shown to facilitate inward-current responses (Dascal & Boton, 1990). Although the data indicate that ionomycin operates mainly via the same mechanism as IP3 in inducing a release of Ca2+ from its intracellular stores, part of the action of ionomycin may be different in nature. It was found that ionomycin could evoke a small inward current following the response evoked by the injection of IP3 (Fig. 6B). In contrast, a second injection of 1P3, instead of the application of ionomycin, did not elicit any appreciable current. This may suggest that the small current evoked by ionomycin is dependent on Ca2+ being released from some Ca2+ stores which are not accessible to IP3, or stores which are insensitive to IP3 (Biden, Wollheim & Schlegel, 1986; Berridge, Cobbold & Cuthbertson, 1988; Thevenod, Dehlinger-Kremer, Kemmer, Christian, Potter & Schulz, 1989). However, an alternative explanation is that 1P3 acts locally inside the cell when injected, i.e. 1P3 cannot effectively act on all intracellular Ca2+ stores because of its limited diffusion in the viscous cytoplasm of the Xenopus oocyte and also dilution. In conclusion, ionomycin is considered to be a useful tool for investigating aspects of the oocyte Ca2+ release mechanism. Combined electrical recordings and direct Ca2+ monitoring would be useful for further study on the mechanism of ionomycin effects on Ca2+ release from the intracellular stores in Xenopus oocytes. We thank Drs K. A. Eidne, P. L. Taylor, M. Yoshimura, Mrs J. Zabavnik and Professors D. W. Lincoln, C. R. House and K. Taniyama for their support, Mr D. G. Doogan for animal care, and Messrs T. E. McFetters, E. W. Pinner and M. Yogata for making the figures. The work was supported by the Medical Research Council (MRC, UK) and S. Plant was an MRC student. REFERENCES
BARRITT, G. J. & LEE, A. M. (1985). Effects of electrical stimulation and an intracellular calcium chelator on calcium movement in suspensions of isolated myocardial muscle cells. Cardiovascular Research 19, 370-377. BERRIDGE, M. J. (1987). Inositol trisphosphates and diacylglycerol: two interacting second messengers. Annual Review of Biochemistry 56, 159-193. BERRIDGE, M. J. (1988). Inositol trisphosphate-induced membrane potential oscillations in Xenopus oocytes. Journal of Physiology 403, 589-599. BERRIDGE, M. J., COBBOLD, P. H. & CUTHBERTSON, K. S. R. (1988). Spatial and temporal aspects of cell signalling. Philosophical Transactions of the Royal Society B 320, 325-343. BERRIDGE, M. J. & IRVINE, R. F. (1989). Inositol phosphates and cell signalling. Nature 341, 197-205. BIDEN, T. J., WOLLHEIM, C. P. & ScHLEGEL, W. (1986). Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Journal of Biological Chemistry 261, 7223-7229. BOTON, R., DASCAL, N., GILLO, B. & LASS, Y. (1989). Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187. Journal of Physiology 408, 511-534. BOYNTON, A. L., COONEY, R. V., HILL, T. D., NILSSON, T., ARKHAMMAR, P. & BERGGREN, P.-O. (1989). Extracellular ATP mobilizes intracellular Ca2" in T51B rat liver epithelial cells: a study involving single cell measurements. Experimental Cell Research 181, 245-255. COHEN-ARMON, M., SOKOLOVSKY, M.& DASCAL, N. (1989). Modulation of the voltage-dependent sodium channel by agents affecting G-proteins: a study in Xenopus oocytes injected with brain RNA. Brain Research 496, 197-203.
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DASCAL, N. (1987). The use of Xenopus of Biochemistry 22, 317-388. DASCAL, N. &
BOTON, R.
oocytes for the study of ion channels. CRC Critical Review
(1990). Interaction between injected
Xenopus oocytes. FEBS Letters 267, 22-24.
Ca2+ and intracellular Ca2+ stores in
DASCAL, N., GiuLo, B. &
LASS, Y. (1985). Role of calcium mobilization in mediation of acetylcholine-evoked chloride currents in Xenopus laevis oocytes. Journal of Physiology 366,
299-313.
DASCAL, N., IFUNE, C., HOPKINS, R., SNUTCH, T. P., LUBBERT, H., DAVIDSON, N., SIMON, M.
I.
&
LESTER, H. A. (1986). Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA. Molecular
Brain Research 1, 201-209. DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in
laboratory maintained animals. Journal of Morphology 136, 153-180. GILLO, B., LASS, Y., NADLER, E. & ORON, Y. (1987). The involvement of inositol 1,4,5trisphosphate and calcium in the two-component response to acetylcholine in Xenopus oocytes.
Journal of Physiology 392, 349-361. LECHLEITER, J., GIRARD, S., CLAPHAM, D. & PERALTA, E. (1991 a). Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors. Nature 350, 505-508. LECHLEITER, J., GIRARD, S., PERALTA, E. & CLAPHAM, D. (1991 b). Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes. Science 252, 123-126. C. M. & HARMANN, T. E. (1978). Characterization of ionomycin as a calcium ionophore. Journal of Biological Chemistry 253, 5892-5896. NOMURA, Y., KANEKO, S., KATO, K., YAMAGISHI, S. & SUGIYAMA, H. (1987). Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTPbinding proteins in Xenopus oocyte after injection of rat brain messenger RNA. Molecular Brain Research 2, 113-123. ORON, Y., DASCAL, N., NADLER, E. & LuPu, M. (1985). Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes. Nature 313, 141-143. PARKER, I. & IVORRA,I. (1990). Localized all-or-none calcium liberation by inositol trisphosphate.
LIu,
Science 250, 977-979. PHILLIPS, D.S. (1978). Basic Statistics for Health Science Students. W. H. Freeman and Company, San Francisco. RANA, R. S. & HOKIN, L. E. (1990). Role of phosphoinositides in transmembrane signaling. Physiological Reviews 70, 115-164. SNUTCH, T. P. (1988). The use of Xenopus oocytes to probe synaptic communication. Trends in Neurosciences 11, 250-256. THEVENOD, F., DEHLINGER-KREMER, M., KEMMER, T. P., CHRISTIAN, A.-L., POTTER, B. V. L. & SCHULZ,I. (1989). Characterisation of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and -insensitive (1isCaP) nonmitochondrial pools in rat pancreatic acinar cells. Journal of Membrane Biology 109, 173-186. TIGYI, G., DYER, D., MATUTE, C. & MILEDI, R. (1990). A serum factor that activates the phosphatidylinositol phosphate signaling system in Xenopus oocytes. Proceedings of the National Academy of Sciences of the USA 87, 1521-1525. TSIEN, R. Y. (1981). A non-destructive technique for loading calcium buffers into cells. Nature 290, 527-528. YOSHIDA, S. & PLANT, 5. (1991). A potassium current evoked by growth hormone-releasing
Ca2+
hormone in follicular oocytes of Xenopus laeVis. Journal of Physiology 443, 651-667. YOSHIDA, S., PLANT, S., TAYLOR, P. L. & EIDNE,, K. A. (1989). Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA. Molecular Endocrinology 3, 1953-1960.
Journal of Physiology (1992), 458, pp. 307-318 With 6 figures Printed in Great Britain
MECHANISM OF RELEASE OF Ca2+ FROM INTRACELLULAR STORES IN RESPONSE TO IONOMYCIN IN OOCYTES OF THE FROG XENOPUS LAEVIS
BY SHIGERU YOSHIDA* AND SUSAN PLANTt From the Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, the Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan and the Department of Preclinical Veterinary Sciences, University of Edinburgh, Summerhall, Edinburgh EH9 1QH (Received 28 August 1991) SUMMARY
1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole-cell currents using the twoelectrode voltage-clamp method. 2. The extracellular application of ionomycin, a selective Ca2+ ionophore, evoked an inward current consisting of a spike-like fast component followed by a long-lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose-dependent manner and was dependent on Cl-. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0 1 ftM). In contrast, a larger ionomycin response (1 ,lM), consisting of fast and slow components, was followed by refractory period. Washing for 50-90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA AM, 1-10 ,tM), a membrane-permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX, 0-3-2-0,tg/ml), a blocker of guanine nucleotide-binding regulatory proteins (G proteins). In contrast, the response to acetylcholine (ACh), which is known to occur via a G protein, was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mm EGTA), whereas the slow component was suppressed. 7. Injection of inositol 1,4,5-trisphosphate (IP3) following a response to extra* Present address: Department of Physiology, Nagasaki University School of Medicine, Nagasaki 852, Japan. t Present address: Department of Physiological Sciences, The Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH.
MS 9672
S. YOSHIDA AND S. PLANT cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast, ionomycin evoked a small inward current when it was applied after an inward-current response evoked by 1P3 injection, whereas a second injection of 1P3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins, resulting in activation of Ca2+-activated Clchannels, (b) ionomycin mainly acts on the same intracellular Ca2+ stores as 'P3, and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response, whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores. 308
INTRODUCTION
Xenopus oocytes have been widely used for functionally expressing receptors and ion channels by injecting exogenous mRNAs isolated from a variety of preparations to determine their amino acid sequences and also for investigating endogenous receptors for acetylcholine (ACh) etc. (see Dascal, 1987; Snutch, 1988 for reviews). In these cases, receptors are coupled to endogenous pathways in Xenopus oocytes, which elicit inward-current responses consisting of two components, a rapid transient and a long-lasting slow component. All these receptor mediated current responses have been found to operate via Ca2+-activated Cl- channels. Stch Clchannels are activated when the concentration of intracellular Ca2+ is increased, mainly by the release of Ca2+ from intracellular stores. Xenopus oocytes have been used as a convenient material for studying the mechanism of Ca2' release. Recently, the quanta4 and spacio-temporal aspects of Ca2+ release in Xenopus oocytes have been studied in highly localized regions using laser confocal microscopy. Local Ca2+ release occurred in an all-or-none mariner in response to photoreleased inositol 1,4,5-trisphosphate (1P3), suggesting that Ca2+ release from larger areas is a collection of quantized local Ca2+ release (Parker & Ivorra, 1990). Examining muscarinic ACh receptors, which are coupled to turnover of phosphatidyl inositol, Lechleiter and his co-workers (Lechleiter, Girard, Peralta & Clapham, 1991b) found regenerative spiral waves of retease of free Ca2+, Further study revealed differences in timing and magnitude of Ca2+ release among subtypes of muscarinic ACh receptors (Lechleiter, Girard, Clapham & Peralta, 1991 a). Although, these studies clarify some aspects of intracellular release of Ca2+, the mechanism of increase in the intracellular concentration of Ca2+ ([Ca2+]i) in response to ligands is still a matter of debate. The action of A23187, a Ca2+ ionophore, has been investigated in Xenopus oocytes (Gillo, Lass, Nadler & Oron, 1987); pretreatment of oocytes with A23187 resulted in depletion of intracellular Ca2+ stores. It has also been reported that A23187 elicited a slow inward current in Xenopus oocytes (Boton, Dascal, Gillo & Lass, 1989). However, A23187 was mainly used to permealize oocytes to Ca2+, and no work was attempted to study the action of A23187 itself on Ca2+. The aim of the present work is to study the mechanism of elevation of [Ca2+]i using another Caa2+ ionophore, ionomycin. Ionomycin is known to release Ca2+ from intracellular stores in various types of cells (Liu & Harmann, 1978; Boynton, Cooney, Hill, Nilsson, Arkhammar & Berggren, 1989). It was found that ionomycin elicited an inward current response,
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consisting of fast and slow components, and mimicked receptor-mediated responses. The ionomycin response was also similar to current responses evoked by the injection of 1P3 (Oron, Dascal, Nadler & Lupu, 1985; Gillo et al. 1987). METHODS
Preparation of Xenopus oocytes. Mature oocytes, at stages V and VI (Dumont, 1972), were collected from the ovary of adult female frogs of Xenopus laevis which were anaesthetized by cooling with ice (Yoshida & Plant, 1991). Oocytes were freed from the connective tissue and follicle cell layer with 2 mg/ml collagenase (Sigma, Type IA) in Ca2+-free saline for 1-5-2-5 h at room temperature (20-23 °C). These 'defolliculated' or 'denuded' oocytes were stored at 19 'C in modified Barth's solution supplemented with penicillin (100 IU/ml), streptomycin (100 lOg/ml), 0-25 -nm sodium pyruvate and 2 % Ficoll (Sigma) before electrophysiological study. Electrophysiology. The conventional two-electrode voltage-clamp method, using a home-made voltage-clamp system, was used to measure whole-cell currents in Xenopus oocytes as described previously (Yoshida, Plant, Taylor & Eidne, 1989; Yoshida & Plant, 1991). DC resistances of membrane potential-recording and current-injection electrodes, filled with 3 M KCl, were 1-3 MQ and 08-1-5 MQ, respectively. Membrane currents were processed by a digital audioprocessor (SONY PCM-701ES; sampling frequency 44-1 kHz) modified to accept down to DC and stored on videotapes with a video cassette recorder (Panasonic NV-G12B). The stored data were displayed on a chart recorder (Graphtee SR6335-2L) for illustrations. The reversal potential for current responses to ionomycin was estimated by the ramp method (Yoshida & Plant, 1991); repetitive voltage ramps, 0-8-1 0 s in duration with slopes of 80-120 mV/s, were applied to an oocyte during a response to ionomycin. Solutions. The ionic composition of the standard solution used for electrophysiology was: 115 mM NaCl, 2 mm KCl, 1-8 mm CaCl2, 1 mM MgCl2 and 10 mm HEPES (pH 7 4). Chloride- was replaced with the membrane-impermeant anion methanesulphonate- (Aldrich), and Na+ was replaced with Tris+ (Sigma). lonomycin (free acid form), a Ca2+ ionophore, was purchased from Calbiochem (LaJolla, CA, USA). The acetoxymethyl ester of bis-(O-aminophenoxy)-ethaneN,N,N',V'-tetraacetic acid (BAPTA AM; Calbiochem), a membrane-permeable intracellular Ca2+ chelator, was used to chelate intracellular Ca2+ (Barritt & Lee, 1985). lonomycin and BAPTA AM were firstly dissolved in dimethyl sulphoxide (DMSO; Sigma); the final concentration of DMSO in the bathing medium was between 0 1 and 0 5 % (v/v). DMSO had no effect on its own at these concentrations. Ethylene glycol bis-(/?-aminoethylether)-N,N,N'N'-tetraacetic acid (EGTA; a Ca21 chelator), inositol 1,4,5-trisphosphate (JP3, potassium salt) was obtained from Sigma. Injection of chemicals into oocytes was carried out using a pressure-injection system (Hamilton Microlab-M, Switzerland). Pertussis toxin (PTX) was obtained from Calbiochem and Sigma. When necessary oocytes were treated with PTX (0-3-2-0 ,ug/ml) for 16-19 h in the standard solution which was supplemented with a stabilizer, bovine blood albumin (0 3 mg/rnl; Wako Pure Chemical Industries, Japan) (Cohen-Armon, Sokolovsky & Dascal, 1989). The pH of all solutions was adjusted to 7 4, and solutions were introduced to and removed from the experimental chamber by perfusion. Statistical analysis. Since responses to chemicals varied from frog to frog (Yoshida & Plant, 1991), oocytes from a single donor were used in the present study to examine the effects of ligands. Also, as the number of observations in the present work was smaller than 30, the t test, one of the parametric tests, was not suitable because it required normal distribution and homogeneity of variance. Therefore, the Mann-Whitney U test (two-tailed with a significance limit of 0-05), one of the non-parametric tests (or distribution-free statistics), was used for the statistical analysis of two independent groups (Phillips, 1978). The Mann-Whitney U test needs only nominal or ordinal scale data and does not require normal distribution or homogeneity of variance. RESULTS
Current responses to ionomycin The resting potential of defolliculated Xenopus oocytes measured with a single voltage-recording electrode in standard solution ranged between -47 and -76 mV, and the average value from representative oocytes was - 618 +8-2 mV (mean +
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A 01 ,uM IM
C
B
5uM IM
1,uM IM
D 05,uM ACh
I100 nA
1000QnA (C)
5 min
Fig. 1. Inward current responses to ionomycin (IM; A-C) and acetylcholine (ACh; D) in defolliculated oocytes of Xenopus laevis. The two-electrode voltage-clamp method was used to record whole-cell currents from the oocyte. The holding potential was -60 mV in all cases. The application of IM and ACh is indicated by horizontal bars. Solutions were introduced to and drained from the experimental chamber by perfusion. The perfusion dead time was less than 2 s in all experiments. Downward deflections indicate inward currents in all figures. The amplitude calibration of 100 nA is for A, B and D, and 1000 nA for C. The time scale applies to all records. A 1 aM ionomycin 30nmn
r60m
B 01 ,uM ionomycin 100 nA 40 nA
(A) (B)
30 min
Fig. 2. Reproducibility of the ionomycin response. A, 1 ,4M ionomycin was repetitively applied to the same oocyte. The application of ionomycin is indicated by horizontal bars. Parallel bars show interval of the record. B, 0 1 4UM ionomycin was repetitively applied to the oocyte as indicated by horizontal bars. Note that the current calibration of 100 nA is for A, and 40 nA for B.
S.E.M.; n = 20). Therefore, oocytes were routinely voltage clamped at -60 mV unless otherwise stated. In defolliculated Xenopus oocytes, ionomycin evokes an inward-current response which is dependent on the release of Ca21 from intracellular stores. A typical example is shown in Fig. 1 A-C. The extracellular application of ionomycin (IM) by perfusion (indicated by horizontal bars) evoked inward-current responses in a dose-dependent manner. With a low concentration of ionomycin the response was a slow inward current (Fig. IA). The response consisted of a spike-like fast component followed by
MECHANISM OF Ca2+ RELEASE FROM STORES
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a smooth slow component when the concentration of ionomycin was increased (Fig. 1 B and C). When the ionomycin concentration was below 1 ,CM, the amplitude of the ionomycin response did not exceed 1000 nA. At higher concentrations of ionomycin, e.g. 5jM, the response could be larger than 1000 nA as shown in Fig. 1 C. The mean values of the peak amplitude of the ionomycin response (the peak amplitude of the fast component when it is obvious) were approximately 30 nA (0 1 /M ionomycin; n = 3), 80 nA (0 5 /SM ionomycin; n = 5), 330 nA (1 /LM; n = 5), and 3010 nA (5 sM; n = 5). The estimated EC50 was approximately 1-7 #M. The ionomycin responses were similar in shape to current responses which are evoked by the activation of receptors like the endogenous ACh receptor for example (Fig. 1D); the application of ACh (horizontal bar) produced an inward-current response which also consisted of two components. The differences are (a) that the ACh response showed current oscillations superimposed on the slow component (this feature is prominent in the oocyte shown in Fig. 4B) and (b) that washing-out of ionomycin from the extracellular perfusate could not abort the ionomycin response (Figs lB and 4A), whereas the ACh response subsided when ACh was removed from the bathing solution (Figs ID and 4B and C). A refractory period followed the ionomycin response when it was large in size and consisted of fast and slow components (n = 7). A representative example is shown in Fig. 2A; the records were taken from the same oocyte. A response was elicited by the first application of 1 ,UM ionomycin. The second application of ionomycin during the slow component failed to evoke any additional current. Jonomycin was washed out from the bathing medium and the oocyte was challenged by ionomycin for a third time, resulting in an incomplete recovery, but the response consisted of fast and slow components. A full recovery of the ionomycin response was obtained by a prolonged wash. Usually, such a full recovery was observed when oocytes were washed for 50-90 min. In contrast, no appreciable refractory period was observed when lower concentrations of ionomycin were used to elicit inward currents. An example is shown in Fig. 2B. Repetitive application of 01 /SM ionomycin with a short interval evoked inward-current responses similar in size and shape. Such responses were small in size and did not show distinct fast and slow components. Ionic dependence of the ionomycin response In order to examine which ions were responsible for responses to ionomycin, the reversal potential for the ionomycin-evoked current was measured using the ramp method (see Methods). The measured value was around -25 mV (- 25-7 + 3-3 mV; n = 3) and was compatible with the reversal potential for Cl- in Xenopus oocytes (Dascal, 1987; Yoshida et al. 1989). Furthermore, a depolarizing shift of approximately 17 mV was observed in the reversal potential of the ionomycin response when the extracellular Cl- concentration was halved (Yoshida et al. 1989). This shift is in good agreement with the expected value of 17-5 mV given by the Nernst equation. The results indicate that the current response evoked by ionomycin is predominantly dependent on Cl-. 11
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Effect of an intracellular Ca2+ chelator BAPTA on the ionomycin response It is established that the Cl- channels, which are responsible for current responses to ACh, serotonin, substance K etc. in Xenopus oocytes, are activated when the concentration of intracellular Ca2+ is increased, i.e. Ca2+-activated Cl- channels A Control
B BAPTA AM
1100 nA 5 min Fig. 3. Effect of extracellularly applied BAPTA AM, a membrane-permeant intracellular Ca2+ chelator, on the current response to ionomycin. The applications of ionomycin are indicated by horizontal bars. A, a control response to 1 /,M ionomycin. B, a response to 1 /M ionomycin in an oocyte which was pretreated with 10/tM BAPTA AM for 2 h. The time and the current calibrations apply to both responses.
(Dascal, 1987; Snutch, 1988). As the response to ionomycin is similar to receptormediated responses and is dependent on Cl-, the same channel would appear to be involved. This possibility was examined by chelating intracellular Ca2` using bis-(Oaminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), a pH-independent chelator specific for Ca2+. The acetoxymethyl ester of BAPTA (BAPTA AM) was used in the present study because it is membrane permeant, and when it penetrates the cell membrane, it is cleaved by cytoplasmic esterases to yield free BAPTA and chelate intracellular free Ca2+; de-esterified BAPTA is membrane impermeant and locked within the cell (Tsien, 1981). Since no significant differences were observed in the chelating effect of intracellular free Ca2+ by extracellularly applied BAPTA AM in the presence or absence of Ca2+ in the bathing medium (Barritt & Lee, 1985), oocytes were treated with BAPTA AM in standard solution. Ionomycin (1 /tM) was applied to oocytes which had been preincubated with 1 or 1O /LM BAPTA AM for 1-3 h. At 1 ,UM, BAPTA AM did not show significant suppression of the ionomycin response (ni = 5; not illustrated). In contrast, when the concentration of BAPTA AM was increased to 10 ,sM, the ionomycin response was markedly reduced (n = 3). The mean values of the peak amplitude of the fast and slow components of the response to 1 ,IM ionomycin in control oocytes not treated with BAPTA AM (see Fig. 3A for an example) were approximately 110 and 30 nA, respectively (n = 3). Figure 3B displays a response to 1 ,UM ionomycin recorded from an oocyte pretreated with 10 /SM BAPTA AM for 2 h; ionomycin evoked a small and slow inward current without appreciable fast current in this case. The mean peak amplitude of the ionomycin response in such BAPTA AM-treated oocytes was 12 nA (n = 3). The oocytes illustrated in A and B were obtained from the same donor frog. Since BAPTA is a Ca2+ chelator and reported to prevent the transient increase in intracellular free Ca2+ (Barritt & Lee, 1985), it is concluded that the ionomycin response is dependent on the rise in the intracellular free Ca2+ concentration.
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Effect of a blocker of G proteins, pertussis toxin (PTX), on the ionomycin response It has been reported that receptor-mediated responses in Xenopus oocytes act via guanine nucleotide-binding regulatory proteins (G proteins) which are sensitive to pertussis toxin (PTX), e.g. acetylcholine and serotonin receptors (Dascal, Ifune, A PTX (2,g/ml) B Control IM ACh
C PTX (03,ug/ml) Ach
1100 nA 5 min
Fig. 4. Effect of pertussis toxin (PTX) on ionomycin (IM)- and acetylcholine (ACh)evoked responses. A, a response to 1 ,UM ionomycin in an oocyte which was pretreated with PTX (2 ,ug/ml, for 18 h). B, a control response to 05 /M ACh. C, a response to 05 #M ACh recorded in an PTX-pretreated oocyte (03 ,ug/ml, for 18 h).
Hopkins, Snutch, Liibbert, Davidson, Simon & Lester, 1986; Nomura, Kaneko, Kato, Yamagishi & Sugiyama, 1987; Cohen-Armon et al. 1989). These receptormediated responses are dependent on the opening of Ca2+-activated Cl- channels. Although pertussis toxin is reported to inhibit receptor-mediated responses, it did not exert any appreciable effect on the ionomycin response; inward-current responses similar in size and shape were recorded from oocytes which were pretreated with 03 ,tg/ml PTX (n = 4; not illustrated) and even with a high concentration (2 /Lg/ml) of PTX (n = 4). The oocyte shown in Fig. 4A was pretreated with 2 ,sg/ml PTX for 18 h. In contrast, pretreatment of oocytes with 03 ,ug/ml PTX was sufficient to suppress the ACh response (Fig. 4C; n = 4) and no appreciable current was observed at 2 gg/ml PTX (n = 3). The results indicate that G proteins are not involved in the ionomycin response. The dependence of the response to ionomycin on extracellular Ca2+ The ionomycin response could be also elicited in Ca2+-free bathing medium as shown in Fig. 5. Extracellular Ca2+ was removed from the bathing medium and any residual Ca2+ was chelated by 2 mm EGTA. Since the oocyte membrane became too leaky to be voltage clamped in this EGTA-containing Ca2+-free solution, divalent cations were used to replace Ca2+. A control response in standard solution (1-8 mm Ca2+) is displayed in Fig. 5A. As shown in Fig. 5B (Mn2+ substituted for Ca2+) and C (Ba2+ substituted for Ca2+), the amplitude of the fast component of the ionomycin-evoked response did not significantly change, although the fast component frequently showed more prominent multiple peaks, in the absence of extracellular Ca2+. The application of 1 /UM ionomycin is indicated by a horizontal bar in each current trace. In contrast, the slow component was significantly reduced in size under Ca2+-free conditions. The aveyage peak amplitudes of the fast and slow components of responses evoked by 1 ,UM ionomycin were 212 and 152 nA (n = 5) in standard 11-2
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solution, respectively, whereas these values were 228 and 17 nA (Mn2+; n = 4), and 204 and 17 nA (Ba2+; n = 4), respectively. There was no significant change in the amplitude of the fast component of the response. On the contrary, the slow component reduction was statistically significant (P = 0-05) when Ca21 was removed A Control
8 Mn2+
C Ba2+
1200nA 5 min
Fig. 5. Current responses evoked by ionomycin under Ca2+-free conditions. A, a control response evoked by 1 /CM ionomycin in standard solution. B, a current response to 1 /,M ionomycin in Ca2+-free solution; the Ca2+ of the standard solution was replaced with an equimolar amount of Mn2+. C, an inward-current response elicited by 1 ,UM ionomycin in bathing medium where Ca2+ was replaced by Ba2+. Both solutions used for B and C contained 2 mm EGTA to chelate residual Ca2+. Horizontal bars indicate the applications of 1 I/M ionomycin.
from the bathing medium. These results indicate that the fast component is not dependent on extracellular Ca2 , whereas the slow component appears to be somewhat dependent on the entry of Ca2+ from the extracellular solution. Interaction between ionomycin and IP3 in inducing Ca2+ release from the stores, The results obtained using an intracellular Ca2+ chelator BAPTA indicate that a current response evoked by ionomycin is dependent on an increase in the concentration of intracellular free Ca21 (see Fig. 3). It has been reported in various types of preparations, including Xenopus oocytes (Gillo et al. 1987; Berridge, 1988), that an intracellular second messenger inositol 1,4,5-trisphosphate (IP3) acts on the intracellular Ca2+ stores, resulting in the release of Ca2+ (for review see Berridge, 1987; Berridge & Irvine, 1989; Rana & Hokin, 1990). Figure 6A shows that the application of ionomycin (5 /tM) evoked an inward current, with a short latency (horizontal bar marked IM), consisting of fast and slow components. A third glass micropipette filled with 10 /tM IP3 solution was inserted into the oocyte during the slow component of the ionomycin response (downward arrowhead). The pipette insertion caused some damage to the oocyte which was reflected by a jump in the holding current. Then, a single pulse of 50 nl of 10 /tM IP3 solution was delivered by the pressure-injection system into the oocyte (horizontal bar marked IP3) following the ionomycin response, but no significant change was observed in the membrane current (n = 4). This failure in evoking a response by injected IP3 is not due to damage caused by the insertion of the third micropipette because oocytes which had not been previously challenged by ionomycin produced an inward-current response upon injection of IP3 delivered through the third electrode. It is suggested that ionomycin acts on the same intracellular Ca2+ store as IP3 and that the response to ionomycin depletes this store of Ca2+.
MECHANISM OF Ca2+ RELEASE FROM STORES
315
In Fig. 6B, a third electrode containing 10 JM IP3 was inserted into an oocyte and the oocyte membrane was allowed to seal for a few minutes. Then 50 nl of 10 #M IP3 solution was injected by pressure into the oocyte (horizontal bar marked IP3), resulting in an inward current consisting of fast transient and slow components. A IM
B IP3 I
IM
1100 nA 5 min
Fig. 6. Interaction of ionomycin (IM) and inositol 1,4,5-trisphosphate (1P3) in inducing current responses. A, an oocyte was challenged with 5 /M ionomycin and produced an inward current consisting of fast and slow components. A third electrode containing IP3 was inserted into the oocyte during the slow phase of the ionomycin response (downward arrowhead) and 50 nl of 10 #M IP3 was injected. B, an oocyte was firstly challenged with IP3 which was delivered through a third electrode followed by the extracellular application of 5 uim ionomycin. The records A and B were obtained from different oocytes.
When ionomycin (5 ,tM) was applied in succession (horizontal bar marked IM), a small inward current was evoked (n = 3). On the contrary, no substantial current was evoked when oocytes were challenged by a second injection of IP3. DISCUSSION
The present work has shown that ionomycin (free acid form), a selective Ca2+ ionophore, evoked an inward-current response consisting of a spike-like fast current followed by a long-lasting slow current. This ionomycin response mimicked receptormediated responses observed in Xenopus oocytes (Fig. 1). The ionomycin response was also similar to the response elicited by the injection of inositol 1 ,4,5-trisphosphate (IP3; Fig. 6B) (Oron et al. 1985; Gillo et al. 1987). Furthermore, the ionomycin response appeared to share the same mechanism as receptor-mediated and IP3induced responses, i.e. ionomycin activated Ca2+-activated Cl- channels via release of Ca2+ from intracellular stores. There are, however, several differences between the ionomycin and receptor-mediated responses. (a) The involvement of IP3 may not be important in the ionomycin response because the ionomycin response showed very few current oscillations; it has been reported that receptor-mediated responses which occur via the phosphoinositide pathway and responses evoked by the injection Qf 1P3 show current oscillations (fluctuations) superimposed on their slow component (e.g. Dascal, Gillo & Lass, 1985; Gillo et al. 1987). (b) G proteins do not seem to be involved in the ionomycin response (Fig. 4). Blood sera from vertebrate species are reported to elicit oscillatory Cl- currents in Xenopus oocytes (Tigyi, Dyer, Matute &
S. YOSHIDA AND S. PLANT Miledi, 1990). In the present study, 0 3 mg/ml bovine blood albumin (Fraction V; Wako Pure Chemical Industries, Japan) was used as a stabilizer (see Dascal et al. 1986; Cohen-Armon et al. 1989) for testing the effect of pertussis toxin (PTX). The possibility that the inhibition of the ACh response was not due to the blockade of G proteins by PTX but by depletion of the intracellular Ca2" stores by albumin can be excluded because (a) the albumin used in the present study did not elicit any currents on its own (n = 5), (b) ionomycin induced inward-current responses in Xenopus oocytes which were treated with albumin and PTX (Fig. 4), and (c) the ACh response was also inhibited by PTX in oocytes not treated with albumin (n = 3) (see Nomura et al. 1987). 316
Mechanisms underlying the fast and the slow components of the ionomycin response Differences were revealed in the properties of the fast and the slow components of the ionomycin response. The fast component was not substarwtially affected by removing Ca2+ from the bathing solution or by replacing extracellular Ca2+ with Ba2+ or Mn2+, whereas the slow component was significantly suppressed (Fig. 5). The data suggest that entry of Ca2+ from outside the oocyte, including Ca2+ from the plasma membrane (see below), considerably contributes to the slow but not to the fast component of the ionomycin response and that the fast component is predominantly dependent on an intracellular Ca2+-releasing mechanism. Moreover, the fast component of the ionomycin response frequently showed multiple peaks (Figs 1B and 5A), and this was potentiated in the absence of external Ca2+ (Fig. 5), whereas the slow component did not show distinct peaks. The multiple peaks of the fast component suggest (a) ionomycin acts on distinct types of Ca2+ stores with time, e.g. superficially and deeply located Ca2+ stores or (b) Ca2+ which is released by ionomycin causes a cascade of Ca2+-induced Ca2' release. When the intracellular Ca2+ was chelated by BAPTA, an intracellular Ca21 chelator, the fast component of the ionomycin response was greatly suppressed leaving a slow current (Fig. 3). It is reported that higher doses of EGTA, another Ca2+ chelator, are necessary to inhibit the slow component of ACh and serotonin responses than to suppress the fast component inXenopus oocytes (Boton et al. 1989). It was proposed by Gillo and his co-workers (1987) that (a) the fast component of such responses represents rapid interaction of Ca2+ with Cl- channels and requires a relatively high Ca2+ concentration, and (b) the slow component proceeds through the activation of some biochemical event which requires a lower Ca2+ concentration. Also, they speculate that the two components may be mediated by distinct Clchannels (Gillo et al. 1987; Boton et al. 1989).
Actions of ionomycin and IP3 on the intracellular Caa2+ stores It has been reported that injection of inositol 1,4,5-trisphosphate (IP3) mimics receptor-mediated current responses in Xenopus oocytes (Oron et al. 1985; Gillo et al. 1987). The present work shows that the response evoked by ionomycin is similar to both receptor-mediated and lP3-elicited responses. The results displayed in Fig. 6 indicate that ionomycin shares a common intracellular Ca2+ release mechanism with IP3, as no substantial current was elicited by IP3 following the ionomycin response (Fig. 6A). It is unlikely that this effect was due to desensitization of Ca2+-gated Cl-
MECHANISM OF Ca2+ RELEASE FROM STORES 317 channels upon the elevation of intracellular free Ca2+ concentration by ionomycin, because repetitive injections of Ca2+ have been shown to facilitate inward-current responses (Dascal & Boton, 1990). Although the data indicate that ionomycin operates mainly via the same mechanism as IP3 in inducing a release of Ca2+ from its intracellular stores, part of the action of ionomycin may be different in nature. It was found that ionomycin could evoke a small inward current following the response evoked by the injection of IP3 (Fig. 6B). In contrast, a second injection of 1P3, instead of the application of ionomycin, did not elicit any appreciable current. This may suggest that the small current evoked by ionomycin is dependent on Ca2+ being released from some Ca2+ stores which are not accessible to IP3, or stores which are insensitive to IP3 (Biden, Wollheim & Schlegel, 1986; Berridge, Cobbold & Cuthbertson, 1988; Thevenod, Dehlinger-Kremer, Kemmer, Christian, Potter & Schulz, 1989). However, an alternative explanation is that 1P3 acts locally inside the cell when injected, i.e. 1P3 cannot effectively act on all intracellular Ca2+ stores because of its limited diffusion in the viscous cytoplasm of the Xenopus oocyte and also dilution. In conclusion, ionomycin is considered to be a useful tool for investigating aspects of the oocyte Ca2+ release mechanism. Combined electrical recordings and direct Ca2+ monitoring would be useful for further study on the mechanism of ionomycin effects on Ca2+ release from the intracellular stores in Xenopus oocytes. We thank Drs K. A. Eidne, P. L. Taylor, M. Yoshimura, Mrs J. Zabavnik and Professors D. W. Lincoln, C. R. House and K. Taniyama for their support, Mr D. G. Doogan for animal care, and Messrs T. E. McFetters, E. W. Pinner and M. Yogata for making the figures. The work was supported by the Medical Research Council (MRC, UK) and S. Plant was an MRC student. REFERENCES
BARRITT, G. J. & LEE, A. M. (1985). Effects of electrical stimulation and an intracellular calcium chelator on calcium movement in suspensions of isolated myocardial muscle cells. Cardiovascular Research 19, 370-377. BERRIDGE, M. J. (1987). Inositol trisphosphates and diacylglycerol: two interacting second messengers. Annual Review of Biochemistry 56, 159-193. BERRIDGE, M. J. (1988). Inositol trisphosphate-induced membrane potential oscillations in Xenopus oocytes. Journal of Physiology 403, 589-599. BERRIDGE, M. J., COBBOLD, P. H. & CUTHBERTSON, K. S. R. (1988). Spatial and temporal aspects of cell signalling. Philosophical Transactions of the Royal Society B 320, 325-343. BERRIDGE, M. J. & IRVINE, R. F. (1989). Inositol phosphates and cell signalling. Nature 341, 197-205. BIDEN, T. J., WOLLHEIM, C. P. & ScHLEGEL, W. (1986). Inositol 1,4,5-trisphosphate and intracellular Ca2+ homeostasis in clonal pituitary cells (GH3). Journal of Biological Chemistry 261, 7223-7229. BOTON, R., DASCAL, N., GILLO, B. & LASS, Y. (1989). Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187. Journal of Physiology 408, 511-534. BOYNTON, A. L., COONEY, R. V., HILL, T. D., NILSSON, T., ARKHAMMAR, P. & BERGGREN, P.-O. (1989). Extracellular ATP mobilizes intracellular Ca2" in T51B rat liver epithelial cells: a study involving single cell measurements. Experimental Cell Research 181, 245-255. COHEN-ARMON, M., SOKOLOVSKY, M.& DASCAL, N. (1989). Modulation of the voltage-dependent sodium channel by agents affecting G-proteins: a study in Xenopus oocytes injected with brain RNA. Brain Research 496, 197-203.
S. YOSHIDA AND S. PLANT
318
DASCAL, N. (1987). The use of Xenopus of Biochemistry 22, 317-388. DASCAL, N. &
BOTON, R.
oocytes for the study of ion channels. CRC Critical Review
(1990). Interaction between injected
Xenopus oocytes. FEBS Letters 267, 22-24.
Ca2+ and intracellular Ca2+ stores in
DASCAL, N., GiuLo, B. &
LASS, Y. (1985). Role of calcium mobilization in mediation of acetylcholine-evoked chloride currents in Xenopus laevis oocytes. Journal of Physiology 366,
299-313.
DASCAL, N., IFUNE, C., HOPKINS, R., SNUTCH, T. P., LUBBERT, H., DAVIDSON, N., SIMON, M.
I.
&
LESTER, H. A. (1986). Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA. Molecular
Brain Research 1, 201-209. DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in
laboratory maintained animals. Journal of Morphology 136, 153-180. GILLO, B., LASS, Y., NADLER, E. & ORON, Y. (1987). The involvement of inositol 1,4,5trisphosphate and calcium in the two-component response to acetylcholine in Xenopus oocytes.
Journal of Physiology 392, 349-361. LECHLEITER, J., GIRARD, S., CLAPHAM, D. & PERALTA, E. (1991 a). Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors. Nature 350, 505-508. LECHLEITER, J., GIRARD, S., PERALTA, E. & CLAPHAM, D. (1991 b). Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes. Science 252, 123-126. C. M. & HARMANN, T. E. (1978). Characterization of ionomycin as a calcium ionophore. Journal of Biological Chemistry 253, 5892-5896. NOMURA, Y., KANEKO, S., KATO, K., YAMAGISHI, S. & SUGIYAMA, H. (1987). Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTPbinding proteins in Xenopus oocyte after injection of rat brain messenger RNA. Molecular Brain Research 2, 113-123. ORON, Y., DASCAL, N., NADLER, E. & LuPu, M. (1985). Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes. Nature 313, 141-143. PARKER, I. & IVORRA,I. (1990). Localized all-or-none calcium liberation by inositol trisphosphate.
LIu,
Science 250, 977-979. PHILLIPS, D.S. (1978). Basic Statistics for Health Science Students. W. H. Freeman and Company, San Francisco. RANA, R. S. & HOKIN, L. E. (1990). Role of phosphoinositides in transmembrane signaling. Physiological Reviews 70, 115-164. SNUTCH, T. P. (1988). The use of Xenopus oocytes to probe synaptic communication. Trends in Neurosciences 11, 250-256. THEVENOD, F., DEHLINGER-KREMER, M., KEMMER, T. P., CHRISTIAN, A.-L., POTTER, B. V. L. & SCHULZ,I. (1989). Characterisation of inositol 1,4,5-trisphosphate-sensitive (IsCaP) and -insensitive (1isCaP) nonmitochondrial pools in rat pancreatic acinar cells. Journal of Membrane Biology 109, 173-186. TIGYI, G., DYER, D., MATUTE, C. & MILEDI, R. (1990). A serum factor that activates the phosphatidylinositol phosphate signaling system in Xenopus oocytes. Proceedings of the National Academy of Sciences of the USA 87, 1521-1525. TSIEN, R. Y. (1981). A non-destructive technique for loading calcium buffers into cells. Nature 290, 527-528. YOSHIDA, S. & PLANT, 5. (1991). A potassium current evoked by growth hormone-releasing
Ca2+
hormone in follicular oocytes of Xenopus laeVis. Journal of Physiology 443, 651-667. YOSHIDA, S., PLANT, S., TAYLOR, P. L. & EIDNE,, K. A. (1989). Chloride channels mediate the response to gonadotropin-releasing hormone (GnRH) in Xenopus oocytes injected with rat anterior pituitary mRNA. Molecular Endocrinology 3, 1953-1960.
