A caffeine- and ryanodine-sensitive Ca2+ store in bullfrog sympathetic neurones modulates effects of Ca2+ entry on [Ca2+]i.

217

Journal of Physiology (1992), 450, pp. 217-246 With 10 figures Printed in Great Britain

A CAFFEINE- AND RYANODINE-SENSITIVE Ca2l STORE IN BULLFROG SYMPATHETIC NEURONES MODULATES EFFECTS OF Ca2l ENTRY

ON

[Ca2+]i

BY D. D. FRIEL* AND R. W. TSIEN From the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305-5426, USA

(Received 10 April 1991) SUMMARY

1. We studied how in changes in cytosolic free Ca2+ concentration ([Ca2+]i) produced by voltage-dependent Ca2+ entry are influenced by a caffeine-sensitive Ca2+ store in bullfrog sympathetic neurones. Ca2+ influx was elicited by K+ depolarization and the store was manipulated with either caffeine or ryanodine. 2. For a time after discharging the store with caffeine and switching to a caffeinefree medium: (a) [Ca2+]i was depressed by up to 40-50 nm below the resting level, (b) caffeine responsiveness was diminished, and (c) brief K+ applications elicited [Ca2+]i responses with slower onset and faster recovery than controls. These effects were more pronounced as the conditioning caffeine concentration was increased over the range 1-30 mm. 3. [Ca2+]i, caffeine and K+ responsiveness recovered in parallel with a half-time of 2 min. Recovery required external Ca2+ and was speeded by increasing the availability of cytosolic Ca2+, suggesting that it reflected replenishment of the store at the expense of cytosolic Ca2 . 4. During recovery, Ca2+ entry stimulated by depolarization had the least effect on [Ca2+]i when the store was filling most rapidly. This suggests that the effect of Ca2+ entry on [Ca2+]i is modified, at least in part, because some of the Ca2+ which enters the cytosol during stimulation is taken up by the store as it refills. 5. Further experiments were carried out to investigate whether the store can also release Ca2+ in response to stimulated Ca2+ entry. In the continued presence of caffeine at a low concentration (1 mM), high K+ elicited a faster and larger [Ca2+]i response compared to controls; at higher concentrations of caffeine (10 and 30 mM) responses were depressed. 6. Ryanodine (1 /tM) reduced the rate at which [Ca2+]i increased with Ca2+ entry, but not to the degree observed after discharging the store. At this concentration, ryanodine completely blocked responses to caffeine but had no detectable effect on Ca2+ channel current or the steady [Ca2+]i level achieved during depolarization. 7. We propose that, depending on its Ca2+ content, the caffeine-sensitive store can either attenuate or potentiate responses to depolarization. When depleted and in the -

* To whom correspondence should be addressed. MS 9287 8-2

D. D. FRIEL AND R. W. TSIEN 218 process of refilling, the store reduces the impact of Ca2+ entry as some of the Ca2+ entering the cytosol during stimulation is captured by the store. When full, the store amplifies the effects of Ca2' entry by releasing Ca2+ (presumably via Ca2+-induced Ca2+ release), an action that is enhanced by caffeine at low concentration; by preventing net Ca2+ exchange between the store and cytosol, ryanodine abolishes both of these effects. 8. Activity-dependent changes in the Ca2+ content of the caffeine-sensitive store, or regulation of its Ca2+ transport activity, could significantly influence both background [Ca2+]i and stimulus-evoked changes in [Ca2+]i. INTRODUCTION

The cytoplasmic free Ca2+ concentration ([Ca2+]i) is important in the control of diverse cellular processes, and changes in [Ca2+]i provide a way for many physiological stimuli to modify the course of intracellular events. Some stimuli trigger changes in [Ca2+]i by altering Ca2+ transport across the surface membrane, while others act by modifying Ca2+ transport between the cytosol and internal stores. It has become clear that in neurones and other cells these pathways for Ca2+ regulation are not independent. For example, the effects of voltage-dependent Ca2+ entry on [Ca2+]i may reflect not only Ca2+ entry across the plasma membrane but also Ca2+ uptake and release by internal stores (Carafoli, 1987; Blaustein, 1988; Tsien & Tsien, 1990). Ca2+ entry through voltage-gated Ca2+ channels has been studied extensively (e.g. Hess, 1990; Tsien & Tsien, 1990) but by comparison, the functional properties and contributions of internal Ca2+ stores are less well understood. A variety of intracellular organelles are known or suspected to accumulate and release Ca2+ (Miller, 1988; Tsien & Tsien, 1990). In neurones, several types of Ca2+ stores have been distinguished on the basis of the compounds that discharge them, including inositol 1,4,5-trisphosphate (1P3) (Higashida & Brown, 1986; Thayer, Perney & Miller, 1988b) and caffeine (Kuba, 1980; Lipscombe, Madison, Poenie, Reuter, Tsien & Tsien, 1988; Thayer, Hirning & Miller, 1988 a). The caffeine-sensitive store has received particular attention, in part because caffeine-induced release of internal Ca2+ implicates Ca2+-induced Ca2+ release (CICR). CICR is a form of Ca2+ transport that has been studied extensively in muscle cells (Endo, Tanaka & Ogawa, 1970; Ford & Podolsky, 1970; for reviews see Fabiato, 1983; Endo, 1985) where it is thought to involve Ca2+-dependent gating of a Ca2+-permeable channel in sarcoplasmic reticulum (SR) (Rousseau, Smith, Henderson & Meissner, 1986; Imagawa, Smith, Coronado & Campbell, 1987). Caffeine appears to act by shifting the Ca2+ sensitivity of this channel to lower concentrations (Nagasaki & Kasai, 1984; Endo, 1985) and by increasing the probability that the channel is open at saturating [Ca2+]i (Rousseau, LaDine, Liu & Meissner, 1988). In neurones, interest in CICR has grown with the recognition that it has the potential to cause regenerative increases in [Ca2+]i during evoked Ca2+ transients and [Ca2+]i oscillations (Kuba, 1980; Smith, MacDermott & Weight, 1983; Lipscombe et al. 1988). In spite of its intriguing properties, the role of the caffeine-sensitive store in Ca2+ homeostasis is not entirely clear. There have been several studies demonstrating caffeine-induced release of internal Ca2+ in neurones. In sympathetic neurones, effects of caffeine have been studied either indirectly by monitoring [Ca2+]i-

[Ca2+]i CONTROL BY A CAFFEIVE-SENSITIVE STORE

219

dependent membrane conductances (Akaike & Sadoshima, 1989), their effects on membrane potential (Kuba & Nishi, 1976; Kuba, 1980) or directly using fluorescent Ca21 indicators (Nohmi, Kuba, Ogura & Kudo, 1988; Lipscombe et al. 1988; Thayer et al. 1988 a; Hernandez-Cruz, Sala & Adams, 1990). In principle, this store could act .ither as a Ca2+ sink or as a Ca2+ source via CICR (McBurney & Neering, 1987). It has been suggested that the caffeine-sensitive store can act as a Ca21 sink (Lipscombe et al. 1988), but a demonstration of Ca2+ uptake by this store and its effects on [Ca2+]1 has not been given. Nor has it been established whether Ca2+-induced Ca2+ release occurs in neurones under physiological conditions. Lipscombe et al. (1988) showed that in the absence of caffeine, [Ca2+]i continues to rise beyond the end of an electrical stimulus, consistent with the operation of CICR. However, a different conclusion was reached by Hernandez-Cruz et al. (1990), who found that while caffeine mobilized Ca2+ in bullfrog sympathetic neurones, [Ca2+]i declined abruptly after a depolarizing stimulus, consistent with there being little or no CICR in the absence of caffeine. Thayer et al. (1988a) argued that if CICR is present in rat sympathetic neurones, it makes only a small contribution to depolarization-induced [Ca2+]i responses. A starting point for our study is the observation that [Ca2+]i elevations produced by membrane depolarization are attenuated if cells have been recently exposed to caffeine (Neering & McBurney, 1984; Lipscombe et al. 1988; Thayer et al. 1988 a). We sought to understand this effect, and found that attenuation paralleled a decrease in the Ca2+ content of the store and occurred only if Ca2+ entry was induced while the store was refilling. To determine whether prior discharge of the store influenced [Ca2+]i responses by increasing net Ca2+ uptake from the cytosol or by decreasing net Ca2+ release from the store, we used ryanodine to prevent both of these processes, choosing a concentration that had no effect on voltage-gated Ca21 channels. Ryanodine pretreatment completely inhibited responses to caffeine and significantly slowed the rise in [Ca2+]i produced by depolarization; however, the attenuation was not nearly as great as that observed following discharge of the store by caffeine. Our results support the idea that the caffeine-sensitive store can influence stimulus-evoked changes in [Ca2+]i in qualitatively different ways depending on whether it is relatively empty or full. Under conditions which promote Ca2+ entry into the cytosol, the store normally behaves as a Ca2+ source, enhancing [Ca2+]i elevations by releasing Ca2+ into the cytosol, evidently through CICR. In contrast, when the store is depleted and in the process of refilling, it accumulates Ca2+ from the cytosol and greatly attenuates [Ca2+]i responses, acting in this case as a Ca2+ sink. It is suggested that any factor which influences the store's Ca2+ content or the Ca2+ sensitivity of CICR could therefore influence stimulus-evoked changes in [Ca2+]i. A preliminary report of this work has appeared (Friel & Tsien, 1990). METHODS

Cell dissociation and primary culture conditions Bullfrogs (Rana catesbeiana) were killed by decapitation and the sympathetic chains were removed and placed in sterile amphibian Ringer solution whose composition was (in mM): 128 NaCl, 2 KCl, 10 glucose, 10 HEPES, pH 7.3 with NaOH. After removing the surrounding connective tissue, the chains were incubated for 40 min at 35 °C in Ringer solution+ collagenase (3 mg/ml, Worthington) and then transferred to Ringer solution+ trypsin (1-5 mg/ml, Sigma, Type III) and incubated for 10 min. After transferring to fresh Ringer solution the chains were

220

D. D. FRIEL AND R. W. TSIEN

triturated with a 2 ml culture pipette until the solution became cloudy. After washing and re-suspending cells in fresh Ringer solution, a drop of the suspension was placed in the centre of a poly-L-lysine (Sigma)-coated coverglass affixed with Sylgard to the bottom of a 60 mm culture dish, covering a 20 mm hole in the dish. After letting cells adhere to the substrate, a 1:1 mixture of Ringer solution (+2mM-CaCl2) and L-15 medium (GIBCO) supplemented with 25,ug/ml ascorbic acid, 25 ,gg/ml glutathione and 0-25 ,ug/ml DMPH4 (Mains & Patterson, 1973) was added and cells were stored at room temperature (18-20 °C) for up to 1 week. Experiments were carried out in cells having a B-cell appearance (- 30-60 pm in diameter). Although more detailed criteria were not used to select cells for study, all the results were qualitatively consistent over the population of cells investigated.

[Ca24]i measurements To measure [Ca2+]1, cells were incubated in their culture dishes for 40 min at room temperature in a solution containing 2 #1 Fura-2 stock solution/ml Ringer solution. The stock solution contained 1 mM-Fura-2 acetoxymethyl ester and 25 % (w/w) pluronic in dimethyl sulphoxide. Cells were then rinsed with Ringer solution and placed on the stage of an inverted microscope (IM 35, Zeiss) and superfused continuously with Ringer solution. Cells were illuminated with light from a 150 W Xe lamp (Muller Electronic-Optics) transmitted by excitation filters (350 and 380 nm, fullwidth at half maximum = 10 nm) mounted in an eight-position filter wheel which rotated at 20-40 Hz. Excitation light was reflected by a dichroic mirror and focused onto the cell being studied by a 40 x objective (Nikon Fluor, NA 1P3). Emitted light passed through a barrier filter and an aperture centred on the cell body and was measured with a photomultiplier tube (PMT, Thorn, OL 30 Series), giving a spatial average of emitted light intensity over the cell soma. Spatial averaging did not distort the time dependence of [Ca2+]i changes described in this paper, since [Ca24]1 changed with time synchronously over the cell body when measured at a rate of up to 1 Hz using [Ca2+]1 imaging techniques (SIT camera: Dage model 66). This validates spatial averaging to monitor changes in [Ca2+]i that occur over seconds (the maximal sample rate described here is 5 Hz). The PMT signals associated with the two excitation sources were separately integrated (Cairn Research Spectrophotometer) to give two analog signals (F350 and F380) which were updated once per revolution of the filter wheel. Measurements were made periodically (3-5 Hz) by digitizing these signals at 2-5 kHz over 12-8 ms intervals and averaging. Results were then stored on a laboratory comnputer. [Ca2+]1 was calculated from F360 and F380 according to Grynkiewicz, Poenie & Tsien (1985) after subtracting the light intensity measured in the absence of cells (F350,bk and F380,bk) and computing the ratio R = (F360-F30,bk)/(F380-F380,bk). The minimal and maximal values of R, Rmin and RmaxW were determined by measuring F350 and F380 for low- and high-Ca2+ calibration solutions made from a KCI internal solution whose composition was (in mM): 115 KCl, 10 HEPES, 5 MgCl2 (Aldrich salts, 99-99 + % pure), 100 #M-Fura-2 free acid. Low-Ca2+ solutions contained 10 mM-EGTA and high-Ca2+ solutions contained 5 mM-CaCl2. pH was adjusted to 7-3 with KOH after all additions were made. F350 and F380 were measured for these solutions and corrected for background light intensity, measured using the same solution except that Fura-2 was omitted. The dissociation constant for Fura-2 was assumed to be 224 nm, while F38O,l.Ca/F380,highcaS Rmin andRmax, which were remeasured after making optical adjustments, were 11-6-12-1, 0-61-0{98 and 11-0-14-7 respectively. In addition to the effect of caffeine on F350 and F380 related to changes in [Ca2+]1 in intact dyeloaded cells, caffeine had a small effect on fluorescence intensity that appeared unrelated to [Ca2+]i. While it was normally difficult to separate this effect from the large changes in F360 and F380 seen with caffeine-induced changes in [Ca2+]1, it could be observed during recovery from caffeine conditioning (see Results) when caffeine responses were relatively small. The [Ca2+]i-independent changes in F36o and F38o occurred as rapidly as cells could be exposed to caffeine and reversed when caffeine was removed. This effect was also observed in the absence of cells: caffeine increased both F350 and F380 and decreased F360/F380 by increasing F880 more than F360. Under the same conditions, caffeine alone was not detectably fluorescent, implicating a physical interaction between caffeine and Fura-2. Similar effects of caffeine on Fura-2 fluorescence have been reported previously (lino, 1989). While the [Ca2+]i-independent effect of caffeine on Fura-2 fluorescence was comparable in size to that associated with the [Ca2+], undershoot (see Results), it could be easily distinguished from it based on the associated changes in Fs60 and F380. Upon removal of caffeine, the [Ca2+]1independent effect led to a small increase in F360/F380 associated with a steady reduction in both F360 and F380, while the undershoot expressed a time-dependent reduction in F350/F380, reflecting a

_,a2+]i C(ONTROL BY A CAFFEINE-SENTSITIVE STORE

2221

decrease in F350 and an increase in F380. Further support for the distinction between these two effects of caffeine came from the observation that in addition to inhibiting caffeine's ability to release internal Ca2", ryanodine prevented the transient reduction in F350 and rise in F380 ([Ca+] undershoot) which normally followed caffeine removal, but not the small steady decrease in both F350 and F38o.

Electrophysiology Simultaneous measurements of [Ca3+Ji and membrane potential (Vm) were made by impaling Fura-2 AM-loaded cells with a high-resistance microelectrode (R 120 MQ) filled with 3 M-KCl. The membrane potential, Vm, was measured with a List EPC-7 patch clamp under current clamp conditions. Under these conditions, cells reliably generated action potentials in response to current injection. Microelectrodes were used to minimize the disruption of [Ca2+]i homeostasis which occurred with whole-cell recording, as indicated by run-down of responsiveness to high K+ and transformation of [Ca 2]i recovery kinetics (D. D. Friel & R. W. Tsien, unpublished). Vm measurements were acquired and stored in parallel with F350 and F380. (a2+ channel current (Ba 2 as the charge carrier) was measured with a List EPC-7 patch clamp under voltage clamp conditions with low-resistance patch pipettes (R 05 MQ)). Cells were exposed to 2 mM-Ca2+ Ringer solution, accessed by whole-cell dialysis with a CsCl-based internal solution (mM: 127 CsCl, 10 HEPES, 5 MgCl2, pH 7-3 with CsOH) and then exposed to an external solution containing (mM): 130 TEA-Cl, 1 BaCl3, 10 HEPES, 10 glucose, pH 7-3 with TEA-OH, supplemented with 1 /tM-tetrodotoxin (TTX). Whole-cell current was filtered at 1 kHz (8-pole Bessel), digitized at 6 kHz and stored on a laboratory computer. -

-

Solution changes Solution changes were made with a system of microcapillaries (Drummond Microcaps) as in Friel & Bean (1988) except that 20,a1 capillaries were used and solution changes were achieved by moving the capillaries to the cell. Cells were uniformly superfused with the desired solutions, and solution changes were rapid, based on the fast and steady change in Vm achieved when cells were exposed to external solutions with elevated [K+] (see Fig. 1). The internal diameter of the capillaries was approximately five times the typical cell diameter. While cells were locally superfused, the recording chamber was also superfused with Ringer solution at 5 ml/min. High-K+ solutions were equivalent to the Ringer solution described above except that a portion of the Na' was replaced by K+. Nominally Ca2"-free solutions contained 0-2 mM-added EGTA, yielding an estimated free [Ca2"] of - 30 nm, based on an apparent Ca-EGTA affinity of 1 1 x 107 M-1 at pH 7-3 and 50 /tM total contaminating Ca. -

Drugs Sources of drugs were: Fura-2 (acetoxymethyl ester and free acid, Molecular Probes), pluronic (BASF Wyandotte), ryanodine (Research Biochemicals Incorporated), TTX (Calbiochem), caffeine (Sigma), dibutyryl cyclic AMP (Sigma), DMPH4 (Calbiochem). Fresh ryanodine stock solutions (100 mM) were made each day in 95% ethanol and then diluted 105-fold in 2 mM-Ca2` Ringer solution. Control experiments showed that 95 x 10-5 % ethanol did not have any detectable effect on either [Ca2+]i or voltage-dependent Ca2" channel current.

Abbreviations DMPH4, 6,7-dimethyl-5,6,7,8-tetrahydropterine; CCCP, carbonyl cyanide m-chlorophenylhydrazone; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. LHRH, luteinizing hormone-releasing hormone. RESULTS

Our main objective was to study how the caffeine-sensitive store affects cytosolic Ca2" levels following Ca2+ entry through voltage-gated Ca2+ channels. This was done by comparing [Ca2+]i elevations produced by depolarization before and after modifying the store. High-K+ depolarization was used to promote voltage-dependent Ca2+ entry, and the store was manipulated with either caffeine or ryanodine. Cells were depolarized with high K+, rather than with whole-cell voltage clamp techniques,


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to minimize the disturbance of Ca"~homeostasis that occurred with intracellular perfusion (see Methods). In the first part of the paper we will describe how depletion of the caffeine-sensitive store leads to attenuation of [Ca2+ ]i responses produced by Ca2+ entry, a case in which the store acts as a Ca2+ sink. In the second part we will C lO mm-Caff

A 50 mm-K+

50 mm-K+

10mmM-Caff 0 Ca 2

0 Ca 2+

1100 nlm

125 mV 20 s

D

B

is Fig. 1. Comparison between the effects of high K+ and caffeine

(Caff) on [Ca2+] and V. and their different dependence on external Ca2+. A shows the effect of 50 mm-K+ on [Ca2+]i (top trace) and V. (bottom trace) in the presence and absence of external Ca2+ (no added Ca2+ with 0-2 mm-EGTA). High K+ depolarizes V., both in the presence and absence of external Ca2+ but has little or no effect on [Ca2+], in the absence of external Ca2+. B illustrates the first response in panel A on an expanded time scale, showing that the change in [Ca2+]i iS slow relative to the change in V., and occurs at constant V.. C shows the effect of 10 mmcaffeine on a different cell in the presence and absence of external Ca2 . Caffeine increases [Ca2+] and hyperpolarizes Vm both in the presence and absence of external Ca2+, indicating that it releases internal Ca2+. D shows the first response in panel C on an expanded time scale. Dotted lines indicate both zero [Ca2+]i and Vm. Solutions contained 2 mm-added MgCl2. Cell B 1OZ (A and B); cell BtI1 J (C and D). i

show that the store can also potentiate [Ca2+]i elevations by releasing Ca 2+ into the cytosol in concert with stimulated Ca2+ entry, in effect acting as a Ca2+ source.

Contrasting effects of high K+ and caffeine on [Ca2±]i and membrane potential Figure 1 shows the effects of high K+ (A and B) and caffeine (C and D) on [Ca2+]i and membrane potential (Vm), monitored simultaneously with Fura-2 and a microelectrode. Panels A and C each compare [Ca2+]i responses in the presence and absence of external Ca2+ , while B and D show the control responses from A and C on

mm-external Ca2+, 50 mmK+poue 2+]i (A, left). Panel B shows that Vm was

an expanded time scale. In the presence of 2 a depolarization and an elevation of [Ca

[Ca2+]i CONTROL BY A CAFFEINE-SENSITIVE STORE

223

depolarized within one sample interval of starting the solution change (240 ms in this case), and remained stable while [K+]o = 50 mM; thus, high K+ effectively clamps Vm. After [K+]. was restored to 2 mm, [Ca2+]i returned to its prestimulation level with a time course that depended on depolarization magnitude and duration, and Vm underwent a transient hyperpolarization, returning to its prestimulation level within 4-5 s. This response was typical: 50 mM-K' depolarized Vm from a resting potential of -699+25 mV (n = 25, mean+S.E.M.) to -21+1-5 mV (n = 21), and [Ca2]i increased from a resting level of 75-6+3-2 nm (n = 139) by 568+ 18-3 nM (n = 96), rising at a maximal rate of 242+15 nM/s (n = 96). When the K+ challenge was repeated in the absence of external Ca2+ (A, right), Vm depolarized to essentially the same level as it did in the presence of external Ca2+, but there was no detectable increase in [Caa2+]i, verifying that the [Ca2+]i elevation produced by high K+ requires Ca2+ entry (e.g. Lipscombe et al. 1988). The after-hyperpolarization observed after restoring external K+ to 2 mm was more prolonged when [Ca2+]i was elevated (A, left) than when [Ca2+]i remained low (A, right), consistent with the participation of a Ca2+-activated K+ channel (Adams, Jones, Pennefather, Brown, Koch & Lancaster, 1986). In contrast to the effects of high K+, caffeine produced a rise in [Ca2+]i that was associated with membrane hyperpolarization and did not require external Ca2+ (Fig. 1C, and D). When caffeine (10 mM) was applied in the presence of external Ca2 , [Ca2+]i climbed at a maximal rate of 470 + 32 nM/s to reach a peak 439 + 20 nm above the resting level (n = 60) before declining in the continued presence of caffeine. [Ca2+]i responses were accompanied by a transient drop in Vm to -82 +5 mV (n = 8) (C, left). After caffeine was removed, [Ca2+]i fell to a level below the prestimulation level. A second application in the absence of external Ca2+ produced a similar (but slightly smaller) rise in [Ca2+]i (C, right), indicating that caffeine released Ca2+ from an internal store. The caffeine-induced hyperpolarization appeared to be Ca2+ dependent, since it was most pronounced at the peak of the [Ca2+]i transient, whether the peak occurred shortly after caffeine was applied, as with the first response in Fig. 1 C, or with a greater delay, as with the second response. These results establish that high K+ and caffeine influence [Ca2+]i in fundamentally different ways: [K+]o elevation produces a sudden depolarization that promotes Ca2+ entry, while caffeine triggers Ca2+ release from an internal store.

Caffeine pretreatment strongly attenuates subsequent [Ca2+]i responses

to membrane depolarization How does caffeine-induced release of internal Ca2+ influence [Ca2+]i elevations produced by depolarization? Figure 2 illustrates the effect of a caffeine challenge on a subsequent response to 50 mM-K+. Panel A shows a control response to K+ followed by a response to a long ( 2 min) application of 10 mM-caffeine. Caffeine produced a rise in [Ca2+]i followed by a biphasic decline towards the prestimulation level or below, despite the continued presence of caffeine. Accompanying the caffeineinduced rise in [Ca2+]i was a transient hyperpolarization, followed by a late depolarization (Kuba & Nishi, 1976). The late depolarization reached -61 mV (-59 + 2-3 mV, n = 3), which was insufficient to activate voltage-gated Ca2+ channels in these cells (Jones & Marks, 1989). The cell was challenged again with high K+ 135 s -


224

after removing caffeine. Under these conditions, [Ca2+]i increased more slowly following depolarization and recovered more quickly with repolarization. The effect of caffeine was reversible, since a much later K+ depolarization (B) elicited a [Ca21]i rise that more closely resembled the control response. The effects of caffeine A 50 mM-K+

10 mM-Caff

50 mM-K+

20 s

1100 nim

125 mVJ B

C 50mM-IC

50mmM-K

Rec Cont Post-Caff

is Fig. 2. Effects of conditioning with caffeine on subsequent high-K+ responses elicited in the absence of caffeine. A shows responses to 50 mM-K+ (left), 10 mM-caffeine (centre), and K+ (right) applied 135 s after removing caffeine (gap = 44 s). While caffeine conditioning did not change the effects of high K+ on Vm, it strongly attenuated the [Ca2+]i response, slowing the onset and speeding the recovery. These effects of caffeine were reversible (B) as shown by a response elicited near the end of the experiment (45 min after the postcaffeine response in A). C compares the control (Cont) and post-caffeine (Post-Caff) responses from A and the final K+ response from B (Rec) after subtracting prestimulation [Ca2+]1 levels. Cell BilB.

pretreatment on the K+-induced [Ca2+]i response are illustrated on an expanded time scale in panel C, showing that the initial rate of change of [Ca2+]i was decreased by roughly one-half in the wake of caffeine exposure. How might pretreatment with caffeine influence subsequent effects of Ca2+ entry on [Ca2+]i ? Some simple explanations can be ruled out immediately. Caffeine did not have a long-term effect on the resting potential nor did it change the membrane depolarization produced by high K+ (e.g. Fig. 2A). Changes in the responsiveness of voltage-dependent Ca2+ channels can also be excluded based on evidence from wholecell voltage clamp recordings. While caffeine has a small inhibitory effect on Ca2+ channel current, it is reversed within a few seconds of removing caffeine (results not shown; Lipscombe et al. 1988; Thayer et al. 1988 a; see also Hughes, Hering & Bolton, 1990). In the experiment illustrated in Fig. 2, caffeine was removed 135 s before the

225 [Ca2+]i CONTROL BY A CAFFEINE-SENSITIVE STORE second K+ depolarization, providing ample time for recovery of ICa before high K+

was reapplied. Additional evidence against prolonged inhibition of ICa as an explanation of the inhibitory effect of caffeine will be presented below (see 'Evidence for CICR in the absence of caffeine'). It is unlikely that caffeine produced these effects by inhibiting cyclic AMP phosphodiesterase and increasing cyclic AMP, since exposing cells to 1 mM-dibutyrylcyclic AMP had little or no effect on [Ca2+]i or on subsequent K+-induced elevations of [Ca2+]i (three cells). Thus, changes in Vm, ICa or intracellular cyclic AMP do not appear to be responsible for the after-effects of caffeine on subsequent depolarizationinduced [Ca2+]i responses. Depression of Ca2+ responses and basal [Ca2+]i involving Ca2+ accumulation by the caffeine-sensitive store One possible explanation for the effects of caffeine pretreatment on responses to high K+ is that caffeine produces a specific change in the caffeine-sensitive store that persists for a period of minutes after caffeine is removed. To explore this possibility, the effect of pretreatment on subsequent responses to caffeine was monitored. After a standard caffeine exposure (which will be referred to in the following as caffeine conditioning), during which [Ca2+]i stabilized near the resting level (as in Fig. 2), caffeine was removed and then reapplied after variable recovery times (Fig. 3A). Immediately after removing caffeine (up arrow), [Ca2+]i fell below the basal level ([Ca2+]i undershoot); a similar effect in cardiac cells has been described by O'Neill & Eisner (1990). Following short recovery times, responses to test caffeine applications (down arrow) were very small, but after longer recovery intervals responses increased in size (Fig. 3B). Recovery proceeded with a half-time of 124+9 s (n = 4) and was accompanied by a regular increase in the initial and maximal rate at which [Ca2+]i increased following re-application of caffeine. The simplest way to account for the after-effects of caffeine conditioning is that depleting the store limits the amount of Ca2+ that can be released by subsequent caffeine challenges. In terms of this explanation, recovery would reflect replenishment of the store. This interpretation is supported by the results of manipulating cytosolic Ca2+ during the recovery in two ways. Figure 4A and B shows that recovery can be slowed by removing external Ca2+. In the presence of 2 mM-Ca2+, 4 min permitted nearly complete recovery (A). However, in the absence of external Ca2+, very little recovery was observed over the same interval (B), while restoring external Ca2+ to 2 mm was followed by complete recovery of caffeine responsiveness (not shown). These results are representative of observations in each of five cells. Figure 4D-F demonstrates that recovery can be facilitated by a stimulus-evoked Ca2+ transient. For this cell, the interval between conditioning and test caffeine applications was only 92 s, which permitted no detectable recovery (D). However, elevating [Ca2+]i with a brief K+ challenge during the recovery period greatly accelerated recovery of responsiveness to caffeine (E), much as stimulated action potentials accelerate recovery in rat dorsal root ganglion cells (Neering & McBurney, 1984). In the absence of external Ca2+, high K+ neither increased [Ca2+]i nor speeded recovery (F). Thus, it was the rise in [Ca2+]i, rather than the depolarization per se, that was responsible for facilitating recovery in D.

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D. D. FRIEL AND R. W. TSIEN These results support the idea that caffeine conditioning affects subsequent responses to caffeine by reducing the Ca2+ content of the caffeine-sensitive store (rather than, for example, by reducing its sensitivity to caffeine), and that recovery of responsiveness reflects replenishment of the store. They also suggest that the store A

100 nM

20s

t

B

-co 4

0

~072 0,

C4 C -

n n u

0

50 100 150 Recovery time (s) Fig. 3. Recovery of caffeine responsiveness after conditioning with caffeine. A illustrates a series of responses elicited by 10 mM-caffeine after incubating in drug-free medium for various periods of time following conditioning. Cells were conditioned by exposing them to caffeine, which produced a transient rise in [Ca2+]i (not shown), and waiting until [Ca2+]1 stabilized near the resting level. Caffeine was then removed (up arrows) and after various recovery times re-applied (down arrows). After caffeine was removed, [Ca2+]i fell below the resting level and then approached its initial value over several minutes (see text). B plots integrated [Ca2+], for responses shown in A as a function of recovery time. [Ca'+], was integrated after subtracting the steady [Ca2+]i level reached by the end of the conditioning period. Following increasing recovery times, caffeine elicited larger responses with graded kinetic features. Cell B080.

refills by accumulating Ca2+ from the cytoplasm and not directly from the extracellular solution. This is based on the dependence of recovery rate on the availability of cytosolic Ca2+ (Fig. 4D-F) and on the perturbation of [Ca2+]i that


227

follows caffeine removal. When caffeine was removed in the presence of 2 mmexternal Ca2+, [Ca2+]i fell transiently below the resting level (Fig. 4C). Removing external Ca2+ lowered resting [Ca2+]i and slowed or largely prevented recovery from the undershoot, just as it impeded recovery of responsiveness to caffeine. In terms of D

[Ca2+]0 =2 mM

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I1100 nlm ..

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Fig. 4. Ca2+ dependence of recovery following conditioning with caffeine. Left column, A-C: recovery of caffeine responsiveness requires external Ca2+. A shows two responses elicited by 10 mM-caffeine separated by 4 min, which in this cell was sufficient to permit almost complete recovery of caffeine responsiveness. In the absence of external Ca2+ (B), caffeine elicited a first response but there was little recovery over the same time interval. After exposing the cell to 2 mM-Ca2 , caffeine elicited a response comparable to that shown in A (not shown). C shows the [Ca2+]i undershoot which occurred after removing caffeine from the same cell, both in the presence and absence of external Ca2 . Cell BlOD. Right column, D-F: recovery of caffeine responsiveness is speeded by increasing [Ca2+]1. D shows the effects on another cell of two caffeine applications separated by a period (92 s) that did not permit recovery. Note that the recovery interval was shorter than that shown in panel A. In E, 50 mM-K+ was applied briefly to the same cell during the recovery period, which greatly facilitated recovery. F, in the absence of external Ca2 , the pattern of recovery is not influenced by K+ and more closely resembles that shown in panel D, suggesting that it is the rise in [Ca2+]1, rather than the depolarization, which facilitates recovery. Cell BIOH.

228


these ideas, removing external Ca2` slows recovery (Fig. 4A and B) by reducing the availability of cytosolic Ca2+. Influence of store repletion on depolarization-induced [Ca2+]i responses These results could be relevant to the attenuation of K+ responses following pretreatment with caffeine (Fig. 2). If depletion of the caffeine-sensitive store is a key factor in the attenuation, then recovery of responsiveness to depolarization should occur as the store refills. Figure 5A shows K+ responses elicited at different times (Tr) after conditioning with caffeine, and Fig. 5B shows caffeine responses elicited after equivalent recovery times in the same cell, providing an assay of the store's Ca2+ content for each Tr. The recovery time courses from Fig. 5A and B are compared in Fig. 5C, showing that recovery of K+ responsiveness (A, half-time of recovery, T1 = 96 s) closely paralleled the recovery of caffeine responsiveness (El, 71 = 87 s). This is seen more clearly by scaling the K+ response recovery data (A) to allow direct comparison with the caffeine results. (Similar results were obtained from another cell where Ti = 116 s for caffeine and T, = 124 s for K+.) Figure 5D shows that the [Ca2+]i undershoot, plotted on the same time scale as in C, also parallels the time course of recovery. These results suggest that depletion of the caffeine-sensitive store is a common factor underlying the effects of caffeine conditioning on subsequent responses to caffeine and high K+, and on cytosolic [Ca2+]i. One possible explanation comes from recognizing that the after-effect of caffeine conditioning occurs only while the store is depleted and in the process of refilling. Caffeine responses are small during this time because releasable Ca2+ is limited, and K+-induced Ca2+ entry has less effect on [Ca2+]i because some of the Ca2+ that enters the cytosol is taken up by the store as it refills. Finally, removal of caffeine depresses [Ca2+]i by favouring Ca2+ uptake from the cytosol to the store. This explanation is consistent with the dependence of recovery rate on the availability of cytosolic Ca2+. Normally recovery is slow because the basal rate of Ca2+ entry is low. Limiting the availability of cytosolic Ca2+ by removing external Ca2+ (as in Fig. 4B) further slows (or prevents) recovery, while recovery is greatly speeded by increasing [Ca2+]i (as in Fig. 4E). While Ca2+ accumulation by the store provides one explanation for the aftereffects of caffeine conditioning on depolarization-induced [Ca2+]i responses, it may not be the whole explanation. An additional possibility, considered in the remainder of the paper, is that the store can also act as a Ca2+ source: net release of Ca2+ from the store might contribute to [Ca2+]i responses under control conditions, and release would be diminished after the store is depleted. This possibility arises because caffeine-induced release of internal Ca2+ is diagnostic for Ca2+-induced Ca2+ release in other systems (see Introduction). However, while several studies have shown that caffeine releases internal Ca2+ in neurones, it is not clear whether it does so by activating endogenous Ca2+ release processes that are capable' of responding to physiological stimuli. We now present evidence that at low concentrations caffeine enhances Ca2+-induced release of internal Ca2+.

[Ca2+]i CONTROL BY A CAFFEINE-SENSITIVE STORE A

B 10 s

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Fig. 5. Comparison between the time course of recovery for K+ and caffeine responsiveness after conditioning with caffeine. A illustrates [Ca2J]i responses from a cell that was conditioned with caffeine as described in Fig. 3 (legend) and then tested with 50 mM-K' after various recovery times (Tr); bar beneath records indicates when caffeine was present. B shows a similar series of responses from the same cell, in this case elicited by caffeine after recovery times that were equivalent to those in A. C compares integrated [Ca2+]i responses elicited by K+ (A) and caffeine (Cl) for each Tr. Filled triangles represent integrated K+ responses after subtracting the initial value and normalizing the result, showing that responsiveness to K+ and caffeine recover with the same time course. Responses were quantified by subtracting baseline [Ca'+]i levels, integrating over the time during which stimuli were present, and then dividing by the integral for the maximal recovery time (Tr = 397 s). For K+ responses, baseline [Ca2+]i levels were defined as the prestimulation [Ca2+]i level. For caffeine responses, it was chosen as the steady [Ca2+]i level measured at the end of conditioning. D shows how [Ca2J1i changed during the longest recovery period studied (397 s, from the series represented by B); dashed line indicates the [Ca2+]J level at the time caffeine was removed to initiate recovery. Cell B09S.

229

~~~D.D. FRIEL AND R. W. TSIEN

230230

Ca2+-induced Ca2+ release can potentiate responses to Ca2+ entry in the presence of

caffeine When cells were exposed to high K' in the continued presence of caffeine, [Ca2] responses were modified in a way that was qualitatively different from the postA 30 mm-K+

30 mm-K+

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1 mm-Gaff

C Post-Gaff

B + Gaff

30 mm-K+

30 mm-K+

C~~~~~~ont

Caff

nm10mM 100 +10 mm-Caff ~

~

~

~

~

af

Fig. 6. Comparison between two actions of caffeine on K+-induced [Ca2+] responses. Panel A shows [Ca2+] responses elicited by K+ (30 mm) before, during and at two different times after exposure to 1 mm-caffeine. Compared to control K+ responses, [Ca2+]i increased more rapidly in the continued presence of caffeine but more slowly following removal of caffeine. Panels B and C show how these effects depend on caffeine concentration. B, in the continued presence of caffeine, the effects of K+ are enhanced' relative to control responses (Cont) at 1 mm but are depressed at 10 and 30 mm-caffeine (30 mm data not shown). Note the early transient component of the response and the absence of any clear effect on the steady [Ca2+], level in the presence of 1 mm-caffeine. After restoring [K+]. to 2 mmx, [Ca2+] recovered with a half-time of 17-6 s in the control and 10-6 and 13-4 s in the presence of 1 and 10 mm-caffeine, respectively. C, when elicited after removing caffeine (arrow), K+ responses were attenuated to a degree that increased with caffeine concentration over the range 1-30 mmi (30 mm data not shown). The half-time of recovery varied from T7i = 14-3 s under control conditions, to 1-= 13-4, 7-9 and 2-3 s after rate also varied with conditioning with 1, 10 and 30 mm~v caffeine, respectively. the duration of the K+ application: after conditioning with 10 mm-caffeine, 711 for [Ca2+]1 recovery was 4-2 s after a 69 s K+ challenge, 8-3 s after a 116 s K+ challenge, compared to 13-0 s following a K+ challenge that was not preceded by caffeine conditioning. Cell B120.

Rtecovery

caffeine effect described above. This difference was most evident when cells were

challenged with KI at a lower concentration (30 mm); at this level, K' produced a slower (maximal rate = 50 +7 nm/s) and smaller (219±+33 nm, n = 10) increase in [Ca2I]i than did 50 mm-K'. Figure 6A compares the two effects of caffeine at a single caffeine concentration (1 mm) while panels B and C show how they vary with caffeine

231 [Ca2+]i CONTROL BY A CAFFEIYE-SENSITIVE STORE concentration. By itself, 1 mM-caffeine produced a transient [Ca2+]i elevation (A, second response) that was smaller (221 + 77 nm, n = 4) but otherwise similar to those elicited by 10 mM-caffeine shown previously. After challenging with K+ in the continued presence of 1 mM-caffeine (A, third response), [Ca2+]i increased more rapidly during the onset and recovered more rapidly with repolarization than in controls (A, first and last responses). This is illustrated more clearly in Fig. 6B, where the responses elicited from the same cell before and during exposure to 1 mM-caffeine are superimposed; similar effects were observed in all thirteen cells exposed to 30 mM-K+ in the presence of either 1 or 5 mM-caffeine. K+ responses elicited in the presence of caffeine at these concentrations also exhibited a clear inflection during the rising phase that was not apparent in control responses but was often seen in responses to caffeine, particularly when the store was partially empty (e.g. Figs 3 and 5). At higher concentrations (10 and 30 mM), caffeine alone produced larger [Ca2+]1 responses (not shown) and depressed responses induced by K+ (Fig. 6B; results with 30 mm not shown). Acceleration of the rate at which [Ca2+]i recovered after repolarization was also less pronounced at higher caffeine concentrations (Fig. 6, legend). Each of caffeine's actions was reversible (e.g. Fig. 6A). The enhancement of responses to 30 mM-K+ by caffeine at a low concentration (1 and 5 mM; 5 mm data not shown) cannot be easily explained by effects of caffeine on surface membrane transport. A steady reduction in the rate of Ca2+ extrusion from the cytosol by caffeine would increase the rate at which [Ca 2+]i rises following depolarization, but it would also slow the recovery, contrary to what was observed. Direct stimulatory effects of caffeine on voltage-dependent Ca2+ channels would also speed the rise in [Ca2+]i, but only inhibitory effects have been described (Lipscombe et al. 1988; Thayer et al. 1988a; Hughes et al. 1990). It is conceivable that 1 mMcaffeine could increase voltage-dependent Ca2+ entry by changing the depolarization produced by 30 mm-K+ (cf. Fig. 2A), but at this concentration caffeine had no detectable effect on the resting potential or the magnitude of Vm in the presence of 30 mM-K+ (- 33-5 + 1-5 mV, n = 2). Based on these considerations, we suggest that K+-induced changes in [Ca2+]i are accelerated in the presence of 1 mM-caffeine because the effect of Ca2+ entry on Ca2+ release is enhanced. The depression of K+induced [Ca2+]i responses observed in the presence of caffeine at high concentrations (Fig. 6B) might involve inhibition of ICa and depression of the steady [Ca2+]i level achieved by depolarization, but this effect was not studied further. Shortly after removing 1 mM-caffeine, responses elicited by 30 mM-K+ were attenuated (Fig. 6A, fourth response), much as responses to 50 mm-K+ were attenuated after conditioning with 10 mM-caffeine (see above). In contrast to the effects of continuous caffeine on K+-induced [Ca2+]i responses, which varied from enhancement at 1 and 5 mm-caffeine to depression at 10 mm-caffeine and above (Fig. 6B), the after-effects of caffeine conditioning changed monotonically with caffeine concentration over the range 1-30 mm (Fig. 6 C; 30 mm data not shown). The [Ca2+]i undershoot seen immediately after removing caffeine (arrow) became deeper, the subsequent rise in [Ca2+]i evoked by depolarization became slower, and the rate at which [Ca2+]j recovered with repolarization increased (see legend). One explanation for the contrasting concentration dependence of the effects of caffeine shown in Fig. 6B and C can be given as follows. As an extension of the


232

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Ryan

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Post-Caff 2s Fig. 7. Effects of ryanodine on [Ca2+]1 elevations produced by high K+ and caffeine. Top panels show control [Ca2tfi responses elicited by 50 mM-K+ (A) and 10 mM-caffeine (B) while central panels show responses to high K+ (C) and caffeine (D) in the presence of 1 ,UMryanodine. K+ responses elicited before and after treatment with ryanodine from panels A and C are superimposed on an expanded time scale in E after subtracting prestimulation [Ca2+]i. Also shown is a response elicited by K+ after conditioning with 10 mM-caffeine but before the cell was exposed to ryanodine. Note that ryanodine abolished caffeine-induced elevations of [Ca2+]i and slowed the rising phase of the response to 50 mM-K+ without changing the steady [Ca2+]i level seen in the presence of high K+, or the time course with which [Ca2+]i recovered with repolarization. In two out of four cells exposed to 50 mM-K+ in the presence of ryanodine, K+ produced a smaller steady [Ca2+]i elevation after exposure to ryanodine than it did initially, but this could not be distinguished from response run-down due to the lack of reversibility of ryanodine's effects. In the remaining cells, ryanodine did not affect the steady [Ca2+]i elevation produced by high K+. Note also that the [Ca2+]1 response elicited in the presence of ryanodine is intermediate between the control response and that recorded after conditioning with caffeine (E). Compare with the effects of ryanodine on responses to 30 mM-K+ in Fig. 9. Cell B08R.

argument presented above, conditioning with caffeine at a higher concentration leaves the store with a smaller Ca2+ content. When caffeine is removed, Ca2+ is then accumulated by the store at a higher rate. Increased Ca2+ accumulation accounts for

233 [Ca2+]i CONTROL BY A CAFFEINE-SENSITIVE STORE the deeper Ca2+ undershoot, and the slower rise in [Ca2+]i produced by voltagedependent Ca2+ entry. The peaked dose dependence of continuous caffeine can also be accounted for by known properties of CICR. The net effect of the presence of caffeine will depend on the balance between two opposing factors: sensitization of CICR and depletion of the store. Caffeine would be expected to shift the threshold for CICR to lower [Ca2+]i, as in muscle cells (Endo, 1985; Klein, Simon & Schneider, 1990). At low concentrations, this would potentiate the rise in [Ca2+]i produced by Ca2+ entry. At higher caffeine concentrations which significantly deplete the store, little Ca2` is available for release, even though the Ca2+ sensitivity of CICR is high, so that [Ca2+]i responses would not be potentiated. While this does not fully explain the diminished size of [Ca2+]i response elicited in the presence of 10-30 mm-caffeine, the main point is that at low concentrations, caffeine enhances [Ca2+]i responses induced by voltage-dependent Ca2+ entry.

Evidence for Ca2+-induced Ca2+ release in the absence of caffeine While these results suggest that Ca2+ can release internal Ca2+ in the presence of caffeine, they do not determine whether Ca2+ release occurs in the absence of caffeine. To study this point, we used ryanodine. This compound binds with high affinity and specificity to Ca2+ release channels from brain (McPherson, Kim, Valdivia, Knudson, Takekura, Franzini-Armstrong, Coronado & Campbell, 1991) and muscle (Imagawa et al. 1987; Lai, Erickson, Rousseau, Liu & Meissner, 1988). Ryanodine has been used widely in the study of CICR (see Sutko, Ito & Kenyon, 1985; Fill & Coronado, 1988) and is thought to inhibit CICR by binding to the release channel and stabilizing an open subconductance state (Imagawa et al. 1987; Rousseau, Smith & Meissner, 1987), preventing net Ca2+ accumulation by the store and thereby release. In neurones, ryanodine may act in a similar manner (Bezprozvanny, Watras & Ehrlich, 1991) or may prevent opening of the Ca2+ release channel (McPherson et al. 1991). Figure 7 shows responses to 50 mm-K+ and 10 mM-caffeine before and after exposure to ryanodine. Ryanodine (1 jam) completely inhibited responses to caffeine (B and D); inhibition developed in a use-dependent fashion and was not reversed for at least 10 min after removing the drug (Rousseau, Smith & Meissner, 1987; Thayer et at. 1988 a). Ryanodine also modified [Ca2+]i responses elicited by 50 mM-K+ (A and C), slowing the response onset without changing the steady [Ca2+]i levels reached during depolarization or the time course of recovery. This is seen more clearly in Fig. 7E, where K+ responses from A and C are compared on an expanded time scale. Ryanodine slowed the rate at which [Ca2+]i increased in response to high-K+-induced Ca2+ entry in each of six cells (four stimulated with 50 mm-K+, two with 30 mi-K+). This occurred without a significant change in resting [Ca2+]i, which remained within 3+7 % of the level measured before applying ryanodine. Panel E also shows a response to 50 mM-K+, elicited shortly after removing caffeine but before applying ryanodine. Note that under these conditions, [Ca2+]i increased even more slowly with depolarization than it did following treatment with ryanodine. This pattern of differences in the rate at which [Ca2+]i increased under the different conditions (control > ryanodine > post-caffeine) was seen consistently in each of four cells tested (see also Fig. 9). The slower K+-induced rise in [Ca2+]i observed in the presence of 1 jam-ryanodine

D. D. FRIEL AND R. W. TASIEN

234

cannot be explained by diminished voltage-dependent Ca2l entry or stimulation of surface membrane Ca2+ pumps. Ryanodine (1 /tM) had no effect on voltagedependent Ca2+ channel current in voltage-clamped neurones (Fig. 8): neither the time course of the current (Fig. 8A) nor the peak whole-cell current-voltage relation A -10 mV -80

ba

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Fig. 8. Insensitivity of whole-cell Ca2+ channel current to 1 /SM-ryanodine. A shows voltage-dependent Ba2+ current elicited at -10 mV from a holding potential of -80 mV before (a and b), during (c) and after (d) exposure to 1 4uM-ryanodine. B plots peak IB, over the time interval from which the recordings shown in panel A were taken, showing that ryanodine did not have any effects that could be distinguished from steady current run-down. Currents were elicited at 0-2 Hz and records are shown after subtraction of currents leak measured by 12 mV hyperpolarizations from the holding potential. [Ba2+].= 1 mm. Cell B12U.

(not shown) was detectably influenced by ryanodine in each of three cells. Steady stimulation of Ca2+ extrusion from the cytosol across the plasma membrane by ryanodine would reduce the rate at which [Ca2+]i increased in response to voltagedependent Ca2+ entry, but it would also speed recovery following repolarization. However, [Ca2+]i recovered no faster following repolarization (50 mM-K+) in the presence of ryanodine than it did following control depolarizations (four of four cells). With a lower K+ concentration (30 mM) that produced smaller [Ca2+]i elevations, recovery was considerably slower in the presence of ryanodine than under control conditions (two of two cells; see Fig. 9D).


235

Constrained effects of an intracellular compartment on stimulus-evoked changes in

[Ca2+]i The preceding results suggest that the caffeine-sensitive store exchanges Ca2+ directly with the cytosol but not with the extracellular medium, and that caffeine and ryanodine influence high-K+-induced [Ca2 ]i responses by specifically modifying Ca2+ exchange between the store and the cytosol without changing transport processes operating at the surface membrane. If these ideas are correct, then the way in which caffeine and ryanodine influence [Ca2+]i responses should conform with two general principles. The first is that treatments which specifically modify Ca2+ transport between an internal compartment and the cytosol should influence the rate at which [Ca2+]i relaxes toward a new steady-state level, but not the steady-state level itself, following a change in surface membrane Ca2+ transport. Consider a cell with an internal compartment that exchanges Ca2+ directly with the cytosol but not with the extracellular medium, which has a fixed Ca2l concentration co. Let cl and c2 represent the concentrations of Ca2+ in the cytosol and the store. Let ijet represent the net Ca2+ flux from compartment i to j. Then for each instant in time t: = + jnet d(t/t=(Jonet(t) dc,(t)/dt 2 (t)) /Vl and dc2(t)/dt =21 where VT and V2 are the cytosolic and store volumes, respectively. In the steady state, dc1/dt = dc2/dt = 0, so that 0 = (Jonlet*+Jlet*)/ V and 0 1= J21/V2, = 0. If JOt* depends only on c0 and where * denotes the steady-state condition. Thus J21* = cl* and transport processes operating at the surface membrane, described by parameters a,4, then (c c*; a, ) = 0 uniquely determines cl in terms of co and the a' , independently of c2*. This is J illustrated by the simple case in which the unidirectional fluxes across the surface membrane are proportional to concentration: Jol = ao0co, J10 = aiocj, where aoi and a1o are constants. Under steady-state conditions 0 = Jolt*= J* -J* = a -al1c*, which gives c*' = (ceC/cO)c0. These conclusions do not depend on how Jolt* is defined as long as it does not depend on c4, and hold for multiple internal compartments as long as they do not exchange Ca2+ directly with the extracellular compartment. 2

The second principle is that drug-induced changes in net Ca2+ fluxes following depolarization and repolarization should be related to one another. Since the store's Ca2+ content is expected to return to the same steady-state level after repolarization, transport between the store and cytosol which occurs with repolarization should compensate for that which occurs with depolarization. Consequently, net Ca2+ fluxes between the store and cytosol have opposite signs during the onset and recovery phases of the response to depolarization. More importantly, conditions which alter Ca2+ transport between the store and cytosol should produce changes in net Ca2+ fluxes during onset and recovery that are also of opposite sign. Figure 9 shows [Ca2+]i responses elicited by 30 mm-K+ from one cell in the presence of t mM-caffeine (A, + Caff), after conditioning with caffeine (B, Post-Caff), and after exposing the cell to 1 ,tM-ryanodine (D, +Ryan). Responses elicited under each condition are shown with a superimposed control response. Compared to the control (A, Cont,), [Ca2+]i changed more rapidly with both depolarization and repolarization

236

D. D. FRIEL AND R. W. TSIEN A

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Fig. 9. Comparison between caffeine- and ryanodine-induced changes in onset and recovery kinetics and effects on steady-state changes in [Ca2 ],. produced by Ca2+ entry in the same cell. Each panel compares a response elicited under a specified condition with a control response, indicated by Cont,, i = 1,2. Test responses are shown in the order in which they were elicited (top to bottom). Panels A-C compare the two effects of caffeine on K+-induced [Ca24]i responses, and panels D and E illustrate effects of ryanodine. A, in the presence of 1 mM-caffeine, [Ca2+], rises faster and reaches a higher level with depolarization and declines more rapidly following repolarization than it does under control conditions (Contl). B, in contrast, when stimulated 11 6 s after removing caffeine (arrow) and initiating a [Ca24]1 undershoot (Post-Caff), [Ca2+]1 rises much more slowly but eventually resembles the control response. C compares a subsequent control response (Cont2) with that shown in panels A and B (Cont,) demonstrating that responses are reproducible. D, after incubating in the presence of l1uM-ryanodine and achieving complete block of responses to 10 mM-caffeine (not shown), K+ responses were slower both in onset and recovery (+ Ryan) compared to the last control response elicited before applying ryanodine (Cont2). E shows that exposing the cell to 10 mM-caffeine for 83 s in the presence of ryanodine, which had no detectable effect on [Ca2+]1, also had little or no effect on a subsequent response to K+. Caffeine removal was followed by K+ depolarization with the same timing as in panel B, with which it can therefore be compared. Similar results were obtained in this cell with a 129 s caffeine application (not shown). Note that ryanodine relieves part of the attenuation seen in the Post-Caff response and abolishes the [Ca2+]1 undershoot which normally follows removal of caffeine. Prestimulation [Ca2+]1 has been subtracted from the records to facilitate comparison. Cell B12P.

237 [Ca2+]i (CO.VTROL BY A CAFFEINE-SE.NSITIVE STORE in the presence of 1 mM-caffeine (A, + Caff) whereas [Ca2+]i increased much more

slowly when K+ was applied shortly after removing caffeine (B, Post-Caff). A subsequent control response (C, Cont2) agreed closely with Cont,. After exposing the cell to 1 1aM-ryanodine and establishing complete inhibition of responses to 10 mMcaffeine (not shown), the cell was challenged again with 30 mM-Ks in the presence of ryanodine (D, + Ryan). The resulting rise in [Ca2+]i was slower than the control (D, Cont2), but not as slow as that which occurred after conditioning with caffeine (B, Post-Caff), in agreement with results obtained with 50 mM-K+ and 10 mM-caffeine (Fig. 7E). However, in contrast to those results, [Ca2+]i recovered much more slowly with repolarization in the presence of ryanodine than it did in control (D, +Ryan). Since the rate at which [Ca21]i changes at an instant in time, d[Ca21]i(t)/dt, is proportional to the net Ca21 flux entering the cytosol at that time, the results in Fig. 9 make it possible to specify how caffeine and ryanodine influence net Ca21 fluxes entering and leaving the cytosol following step changes in Vm. Comparing d[Ca2+]i(t)/dt at fixed [Ca2+]i before and after treatment with caffeine or ryanodine indicates that: (a) 1 mM-caffeine promotes net Ca2+ flux into the cytosol during depolarization and from the cytosol after repolarization (Fig. 9A); (b) depleting the store reduces net Ca2+ flux into the cytosol following depolarization without affecting net Ca2+ flux from the cytosol following repolarization, as long as [Ca2+]i has achieved a steady level (Fig. 9B); (c) 1 /aM-ryanodine slows both net cytosolic Ca2+ entry upon depolarization and net Ca2+ loss following repolarization (Fig. 9D). The effects of caffeine and ryanodine are completely consistent with the principles presented above. Neither caffeine nor ryanodine influenced the steady-state [Ca2+]i level achieved in the presence of 30 mM-K+. Moreover, in the continued presence of either 1 mM-caffeine or 1 jtM-ryanodine, changes in net Ca2+ fluxes accompanying depolarization and repolarization were of opposite sign. In the presence of caffeine, the net flux during the onset was larger and that during recovery was smaller (i.e. more negative, defining net flux into the cytosol as positive) than the control. In the presence of ryanodine, the net flux was smaller during the onset and larger (i.e. less negative) during recovery, compared to the control. This reciprocal relationship between changes in onset and recovery is not expected for post-caffeine responses (B, Post-Caff) because after conditioning with caffeine the cell was far from steady state. The results in Fig. 9 also provide additional support for our explanation of the after-effects of conditioning with caffeine (e.g. Fig. 9B). In the presence of ryanodine, caffeine had little or no effect on [Ca2+]i (not shown); after removing caffeine (E, arrow) there was no [Ca2+]i undershoot and responses to high K+ were not appreciably different from those elicited before conditioning. This shows that postcaffeine attenuation of high-K+ responses is partially relieved by ryanodine (compare Post-Caff responses in B and E), the expected result if attenuation reflects enhanced net Ca21 uptake by a store that is functionally eliminated by ryanodine. In the Discussion, we will interpret these effects of caffeine and ryanodine in terms of specific drug-induced changes in Ca2+ fluxes between the cytosol and the caffeinesensitive store.


238

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0

Ryanodine ()

+Ryanodine post-Caff Fig. 10. Summary of the effects of caffeine and ryanodine on K+-induced [Ca2+]1 responses in bullfrog sympathetic neurones. Top panel compares a control [Ca2+]i response elicited by 30 mM-K+ (a) with test responses elicited under four different experimental conditions (b-e) from Fig. 9. The bottom part attempts to explain why the test responses differ from the control in terms of caffeine- and ryanodine-induced changes in Ca2+ transport between the caffeine-sensitive store and the cytosol. Arrows indicate direction and magnitude of the net Ca2+ flux across the surface membrane and between the cytosol and the store, during the onset and recovery phases of the response. Under control conditions (bottom, a) depolarization stimulates Ca2+ entry and a rise in [Ca2+]1, which promotes Ca2+ release from the store (onset). Repolarization permits the Ca2+ contents of both the cytosol and the store to relax towards their prestimulation levels (recovery); since the store released Ca2+ during the onset, it must accumulate Ca2+ during the recovery. b, when continuously present, caffeine (1 mM) enhances Ca2+-induced Ca2+ release from the store (onset) so that [Ca2+]1 increases more rapidly than it does under control conditions. To restore its initial Ca2+ content, the store also accumulates Ca2+ more avidly during the recovery; as a result, [Ca2+] declines more rapidly following repolarization (recovery). If Ca2+ entry is induced while the store is filling as in c, net Ca2+ uptake by the store slows the rate at which [Ca2+]i rises during the onset. However, if [Ca2+]i is elevated long enough for the Ca2+ content of both the cytosol and the store to approximate the steady-state values achieved under control conditions (a), then [Ca2+]i will recover just as it does in the control (compare a and c, recovery). By increasing passive Ca2+ exchange between the store and cytosol, ryanodine prevents net Ca2+ accumulation by the store (d and e). Without the added effect of CICR, [Ca2+] rises more slowly in response to stimulated Ca2+ entry (compare a and d, onset) and falls more slowly during recovery (compare a and d) than it does under control conditions. Since the store remains discharged in the presence of ryanodine, caffeine has no further effect. Therefore, the post-caffeine response in the presence of ryanodine (e) is essentially the same as the response elicited in the presence of ryanodine without caffeine pretreatment (d).


239

DISCUSSION

The main conclusion of this paper is that the caffeine-sensitive store can strongly modify the ability of voltage-dependent Ca2+ entry to elevate [Ca2+]i in bullfrog sympathetic neurones. This store may either attenuate or amplify the effects of stimulated Ca2' entry, depending on whether it accumulates or releases Ca2". Ryanodine, a specific antagonist of CICR, inhibited caffeine-induced Ca2+ release and slowed [Ca2+]i elevations produced by K+ depolarization without affecting voltagedependent Ca2+ channel current, providing good evidence for CICR in both caffeineand high-K+-induced [Ca2+]i responses. After discharging the store with caffeine, [Ca2+]i responses were slowed to an even greater extent, indicating that net Ca2+ accumulation by the store can be even more influential than CICR. Overall, these results suggest that the caffeine-sensitive store will influence [Ca2+]i responses in a way that depends on its Ca2+ content and the Ca2+ sensitivity of CICR.

Attenuation and potentiation of depolarization-induced changes in [Ca2+]i by the caffeine-sensitive store Our results can be explained by the simple model shown in Fig. 10 which includes a single internal store that exchanges Ca2+ with the cytosol by means of a ryanodineand caffeine-sensitive pathway. The top part summarizes the different patterns of response to depolarization observed in the absence and presence of caffeine and ryanodine, using individual traces from Fig. 9. The bottom part gives an interpretation of how these patterns arise from drug-induced changes in net Ca2+ fluxes entering the cytosol. Net Ca2+ fluxes across the surface membrane and the store membrane are represented by arrows indicating direction and relative magnitude; net flux into the cytosol is regarded as positive. We distinguish between the onset of the response to depolarization (left column) and the recovery after repolarization (right column). The effects of caffeine and ryanodine are interpreted solely in terms of changes in Ca21 transport between the cytosol and the Ca2+ store: thus, depolarization-induced Ca21 entry during the onset is represented by an arrow of fixed length, as is Ca2+ extrusion across the plasma membrane during recovery. Our interpretation of the effects of caffeine and ryanodine described in this paper are given below: Under control conditions, release of internal Ca2+ through CICR (Fig. 10a, onset) complements net Ca2+ entry stimulated by high K+. During continued depolarization, [Ca2+]i relaxes toward a new steady level determined by the K+-induced increase in Ca2+ permeability of the surface membrane (see below). Following repolarization (a, recovery), Ca2+ is extruded across the plasma membrane, and the store retrieves an amount of Ca2+ equivalent to that which it released during the onset; [Ca2+]i relaxes toward its prestimulation level at a rate determined by the combined effects of these transport processes. In the presence of 1 mM-caffeine, the Ca2+ sensitivity of CICR is increased and depolarization produces a faster rise in [Ca2+]i because release of internal Ca2+ is enhanced (Fig. 10b, onset). As in the control response, [Ca2+]i recovers with repolarization at a rate that reflects both Ca2+ extrusion and Ca2+ uptake. But enhanced release during the onset is matched by enhanced uptake during recovery


240

(b, recovery), so that [Ca2+]i declines more rapidly under these conditions than it does in the control. After depleting the store with caffeine, net Ca2+ uptake from the cytosol perturbs undershoot (Fig. lOc). If Ca2+ entry is stimulated while the causing a store is refilling, part of the Ca2+ which enters the cytosol is taken up by the store. Since this effectively reduces the net flux entering the cytosol, it reduces the rate at which [Ca2+]i increases (c, onset). While [Ca2+]i is elevated, the store fills rapidly (more rapidly than if [Ca2+]i is low; see Fig. 4D-F), permitting [Ca2+]i to approach the steady level achieved with depolarization under control conditions (a). At the instant of repolarization, both [Ca2+]i and the store's Ca2+ content are the same as they were under control conditions, so that [Ca2+]i recovers with essentially the same time course as it did in a. Ryanodine greatly increases passive Ca2+ exchange between the store and cytosol, preventing net Ca2+ accumulation by the store (Fig. lOd and e). As a result, the store no longer influences the way that [Ca2+]i changes in response to Ca2+ entry. Without the added effect of CICR, [Ca2+]i relaxes more slowly than it did under control conditions, both with depolarization (d, onset) and repolarization (d, recovery). Also, since the store is already discharged, caffeine has no effect, and conditioning with caffeine no longer promotes net Ca2+ uptake by the store: in the presence of ryanodine, there is no [Ca2+]i undershoot (compare c and e) and Ca2+ entry has essentially the same effect on [Ca2+]i that it had before conditioning with caffeine (compare d and e). Thus, ryanodine effectively relieves part of the attenuation normally seen after conditioning with caffeine (compare c and e, see below). Figure 10 illustrates how changing the magnitude and sign of the net Ca2` flux from the store into the cytosol (bottom) can produce graded changes in [Ca2+]i responses (top). The initial rise in [Ca2+]i during the onset becomes slower as the net flux from the store declines, from a large positive value in the presence of 1 mmcaffeine (b), to a small positive one in the control (a), reaching zero in the presence of ryanodine (d) and finally becoming negative when the store accumulates Ca2+ as in c. Viewed in this way, attenuation of [Ca2+]i responses after conditioning with caffeine is the consequence of two factors: elimination of Ca2+ release (accounting for the effects of ryanodine; compare Fig. lOa and d) and stimulation of Ca2+ uptake (responsible for the further effect of depleting the store; compare Fig. IOd and c). The next sections address which Ca2+ store(s) participate in these phenomena and the nature of their communication with the cytosol.

[Ca2+]i,

[Ca2+]i

Are otherCa2+ stores involved in the observed effects of caffeine and ryanodine? In Fig. 10 and elsewhere in this paper, we refer to the caffeine-sensitive store as if it were the only store involved. Peripheral neurones contain multiple types of Ca2+ stores whose possible contributions must also be considered. At least two other neuronal Ca2+ stores have been distinguished based on their pharmacological sensitivity: (1) an IP3-sensitive store and (2) a store (presumably mitochondrial) that is sensitive to protonophores such as CCCP and FCCP (Thayer & Miller, 1990; Friel & Tsien, 1990). TheIP3-sensitive store does not appear to be very prominent in amphibian sympathetic neurones, judging from the small [Ca2+]i responses that can

be elicited by agonists that stimulateIP3 production, such as muscarine, LHRH and

substance P (Pfaffinger, Liebowitz, Subers, Nathanson, Almers & Hille, 1988; D. D.


241

Friel, unpublished; see also Adams et al. 1986), and evidence from sensory neurones suggests that the lP3-sensitive store is not influenced by caffeine or ryanodine (Thayer et al. 1988 b). In another study we have shown that the putative mitochondrial store is responsible for the plateau and delayed recovery of [Ca2+]i which follows large elevations of [Ca2+]i elicited by 50 (but not 30) mM-K+ (e.g. see Figs 2 and 7; Friel & Tsien, 1990); domination of the [Ca2+]i recovery kinetics by this store may explain why ryanodine had little effect on the recovery following challenges with 50 compared to 30 mM-K+. While this store is sensitive to FCCP, it is insensitive to ryanodine and caffeine (Friel & Tsien, 1990). The FCCP-sensitive store is apparently not recruited by moderate [Ca2+]i elevations which have prominent caffeine- and ryanodine-sensitive features (D. D. Friel & R. W. Tsien, unpublished observations); thus, the conclusions presented in this paper are probably not significantly influenced by this store. For the purposes of this paper, the caffeine-sensitive store has been operationally defined in terms of its pharmacology, without reference to the intracellular distribution of the caffeine- and ryanodine-sensitive Ca2+ transport process. Our interpretations do not depend on whether the transport process is localized to a single type of Ca2+ store or is distributed among distinct organelles. The key postulate is that caffeine and ryanodine affect the same Ca2+ transport system, i.e. CICR. This is discussed further below. The cytosolic compartment mediates communication between the Ca2+ store and external solution Our results suggest that the caffeine-sensitive store is insulated from the extracellular medium, communicating directly with the cytosol but only indirectly with the external solution. Experimental support for this idea is summarized below: (1) Perturbation of cytosolic calcium following caffeine removal. Replenishment of the store is accompanied by a depression in [Ca2+]i having several characteristics that are expected if it refills at the expense of cytosolic Ca2+: (i) the depth of the [Ca2+]i undershoot varies with the degree to which the store is depleted (Fig. 6C), (ii) the decay of the undershoot depends upon Ca2+ entry from the external solution (Fig. 4C), and (iii) the time course of the undershoot parallels the time course with which the store refills (Fig. 5C and D). (2) Dependence of the rate at which the store refills on the cytosolic calcium level. Recovery of responsiveness to caffeine is slowed or prevented by removing external Ca2+, and greatly speeded by increasing [Ca2+]i (Fig. 4E). This was also found for recovery of responsiveness to high K+ (results not shown). (3) Selective changes in kinetics but not in steady-state calcium level. Caffeine and ryanodine influenced the rate at which [Ca2+]i relaxed in response to a step change in Vm but did not influence the resulting steady-state change in [Ca2+]i (Fig. 9A and D). (4) Linkage between drug-induced changes in net calcium fluxes during depolarization and repolarization. Both caffeine, when present continuously at 1 mm, and ryanodine had opposite effects on net cytosolic Ca2+ fluxes following depolarization and repolarization (Fig. 9A and D). Overall, these results emphasize the ability of the store to influence the time course of cytoplasmic Ca2+ transients initiated by membrane depolarization and repolarization. The next sections summarize evidence for specific effects of ryanodine

242


and caffeine on CICR in these neurones, and compare these actions with those found in isolated systems. Evidence for Ca2"-induced Ca2+ release in the absence of caffeine The results support the hypothesis that CICR can amplify the effect of surface membrane Ca2+ entry under physiological conditions. The key observation is that depolarization-induced Caa2' entry produced a slower rise in [Ca21]i in the presence of ryanodine (Figs 7E and 9D). Our conclusion depends critically on the idea that ryanodine prevents Ca2+ release from the caffeine-sensitive store, without affecting Ca2+ transport across the surface membrane. This idea is based on previous studies of Ca2+ release channels from muscle sarcoplasmic reticulum (see Introduction), which are partially but tonically activated by ryanodine to create a steady Ca2+ leak that empties the store and prevents CICR, and from brain which respond similarly (Bezprozvanny et al. 1991) or are blocked by ryanodine (McPherson et al. 1991). We obtained independent corroboration for this postulated mechanism of action in bullfrog sympathetic neurones. Ryanodine completely abolished caffeine-induced release of internal Ca2+, and modified responses to depolarization in a way that supports a specific action on CICR; this is based on the following considerations. Ryanodine had two effects on high-K+-induced changes in [Ca2+]i: it reduced both the net Ca2+ flux entering the cytosol with depolarization, and the net Ca2+ flux leaving the cytosol after repolarization (Fig. 9D). Insight into these effects can be gained by considering d[Ca2+]i/dt, which provides information about the total net Ca2+ flux (Wtotai) entering the cytosol at each instant in time. Jtotal can be separated into the net flux entering from the extracellular medium and the net flux entering from the store (see above for definition of symbols):

Vld[Ca2 ]i/dt

+ J21t Jtotal = Drug-induced changes in d[Ca2+]i/dt evaluated at fixed [Ca2+]i may be attributed to modified transport processes. In principle, ryanodine could slow the K+-induced rise in [Ca2+]i by modifying either Jonlt or Jn2lt. Several observations argue against changes in surface membrane transport processes. Each net flux JRijet is the difference between the unidirectional flux Jij from compartment i to j, and the unidirectional flux J from j to i: Vld[Ca2+]i/dt = (J1- J1) + (W21 -J12) Ryanodine did not detectably influence the size or kinetics of Ba2+ currents through voltage-dependent Ca2+ channels (Fig. 8; see Valdivia & Coronado, 1989). If these channels represent the main Ca2+ permeability underlying Jo,, then changes in Jo, cannot account for the reduction in d[Ca2+]i/dt observed during the response onset (Fig. 9D). Specific effects on Ca2+ extrusion across the plasma membrane are also unlikely. Steady stimulation of Ca2+ pumps in the surface membrane would increase Jlo and slow the K+-induced rise in [Ca2+]i, but would also depress the steady-state [Ca2+]i level and speed the recovery, contrary to what was observed. This means that if ryanodine increases Jlo it must do so only transiently. But a transient effect on Jlo cannot explain the slower [Ca2+]i recovery observed just after repolarization, apparently expressing a steady-state effect of ryanodine. Thus, it is difficult to account for the observed effects of ryanodine solely in terms of direct modifications =

O

243 [Ca2+]i CONTROL BY A CAFFEINE-SENSITIVE STORE of surface membrane Ca2+ transport processes (although Jo, and J10 are undoubtedly influenced secondarily through changes in [Ca2+]i). A simple explanation for slowing of the response onset is that ryanodine specifically reduces the net Ca2+ flux (J2lt)

from the store to the cytosol (Fig. lOd), either by reducing J21 or increasing J12 While .our results do not distinguish between effects on J21 and J12, previous studies suggest that ryanodine reduces J21 by depleting the store (but see McPherson et al. 1991). This is supported by the observation that caffeine releases internal Ca2+ (i.e. caffeine increases J21) and ryanodine inhibits caffeine-induced release of internal Ca2+. The slowing of recovery could reflect diminished net uptake by the store (less negative Jn2et) due to either an increase in J21 or a decrease in J12.

Effects of modifying the Ca2+ sensitivity of Ca2+-induced Ca2+ release When caffeine was continuously present at 1 mm, high K+ produced a faster and larger increase in [Ca2+]i, reflecting a larger net Ca2+ flux into the cytosol. We attribute this to enhancement of CICR for the following reasons. Arguing as with ryanodine, caffeine could speed the K+-induced rise in [Ca2+]i by modifying surface membrane transport processes, either by increasing Jo, or decreasing Jlo. Enhancement of voltage-dependent Ca2+ entry (Jo,) is unlikely since caffeine is known to inhibit ICa (Lipscombe et al. 1988; Hughes et al. 1990; Sham, Cleemann & Morad, 1991); (inhibition of ICa by caffeine may contribute to smaller [Ca2+]i responses observed in the continued presence of 10 and 30 mm-caffeine). Sustained inhibition of surface membrane Ca2+ pumps would decrease Jlo and speed the K+-induced rise in [Ca2+]i, but would also elevate the steady-state [Ca2+]i level and slow the recovery, in contrast to what was observed. Therefore, if caffeine slows Ca2+ extrusion, it must do so transiently. This conflicts with the observed steady-state effect of caffeine on Ca2+ transport: at the instant of repolarization d[Ca2+]i/dt was much more negative in the presence of caffeine than in the control (Fig. 9A), even though [Ca2+]i was unchanged. Therefore, it is difficult to account for the observed effects of 1 mMcaffeine only in terms of changes in surface membrane Ca2+ transport (although it is likely that Jo, and J1o are influenced by changes in [Ca2+]i). A simple explanation of these observations is that at a low concentration, caffeine influences the net Ca2+ flux (J21lt) between the cytosol and an internal store (Fig. lOb). As with the effects of ryanodine, we cannot distinguish between changes in J21 and J12, but based on previous studies it would be expected that caffeine enhances J21. This is consistent with caffeine's known ability to increase the Ca2+ sensitivity of CICR in muscle cells (Endo, 1985; Klein et al. 1990) and release internal Ca21 in sympathetic neurones. The faster recovery observed with repolarization could reflect enhanced net uptake by the store (more negative Jn2et) due to either smaller J21 or greater J12*

Effects of modifying the Ca2+ content of the caffeine-sensitive store The after-effects of caffeine conditioning were striking: [Ca2+]i was transiently depressed below its basal level, and K+-induced [Ca2+]i responses were greatly attenuated, reflecting diminished net Ca2+ flux into the cytosol compared to control (compare Fig. 9B, Cont, and Post-Caff). We attribute this to diminished Ca2+ influx into the cytosol from the store, due to depletion of store Ca2 , which unmasks an

244


underlying Ca2+ uptake process. This conclusion is supported by the observation that attenuation occurs while the store is refilling and closely parallels the time course of refilling (Fig. 5). The attenuation observed after treatment with caffeine (Fig. 9B) cannot be explained by inhibition of voltage-dependent Ca2+ entry. Direct effects of caffeine on ICa decay within a few seconds after removal of caffeine, whereas attenuation lingers for minutes. Moreover, an explanation in terms of caffeine-induced inhibition of ICa would require that ryanodine somehow relieve this inhibition. On the other hand, the finding that ryanodine prevents the after-effects of caffeine conditioning is expected if ryanodine interferes with caffeine's ability to release Ca2+ from the store. Conditioning with caffeine does not slow K+-induced [Ca2+]i responses by promoting Ca2+-dependent inactivation of Ica, since increasing [Ca2+]i by depolarization promotes, rather than hinders, recovery of responsiveness to high K+ (see above).

Functional implications of Ca2+ transport by the caffeine-sensitive store Our results suggest that the caffeine-sensitive store could either speed (by CICR) or slow (by Ca2+ accumulation) stimulus-evoked changes in [Ca2+]i under physiological conditions, and thereby change the effectiveness of stimuli that control Ca2+_ dependent processes through changes in [Ca2+~]i. Although this paper has focused on modifications of responses to voltage-dependent Ca2+ entry, similar effects are also predicted for changes in [Ca2+]i produced by other stimuli such as 1P3. Stimulusinduced changes in [Ca2+]i could be influenced by any factor which modifies the store's Ca2+ content or the Ca2+ sensitivity of CICR. For example, if the store's Ca2+ content depended on the time average of [Ca2+]i, then subsequent responses to depolarization could be modified in a way that depends on the cell's history of stimulation. Ca2+ sensitivity of CICR is another potential target for regulation. Since depolarization-induced Ca2+ flux in the absence of drugs lies between that observed in the continued presence of a low dose of caffeine, and that observed with ryanodine, it appears that CICR operates well below its full capacity under control conditions. This leaves ample opportunity for positive modulation of CICR. In this regard, it is interesting to ask if endogenous substances, e.g. adenine nucleotides (Endo, 1985), or cyclic ADP-ribose (Galione, Lee & Busa, 1991) might regulate the Ca2+ sensitivity of CICR under physiological conditions. Our results suggest that CICR should influence the kinetics with which [Ca2+]i changes following stimulation but not the steady [Ca2+]i level achieved by long depolarizations; this may be relevant for responses to physiological stimuli (Sah & McLachlan, 1991), which are in the order of 104-105 times faster than the time required to achieve a [Ca2+]i steady state in these cells (see Fig. 9). The authors would like to thank Drs Julie Kauer, Richard Lewis, Antonio Malgaroli and Timothy Ryan for reading and commenting on the manuscript. REFERENCES

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Basal Ca2+ and the oscillation of Ca2+ in caffeine-treated bullfrog sympathetic neurones.

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes.

Store-operated Ca2+ entry in muscle physiology and diseases.

Store-operated Ca2+ entry regulates Ca2+-activated chloride channels and eccrine sweat gland function.

Molecular Determinants for STIM1 Activation During Store- Operated Ca2+ Entry.

Store-Operated Ca2+ Entry Sustains the Fertilization Ca2+ Signal in Pig Eggs.

Mid-range Ca2+ signalling mediated by functional coupling between store-operated Ca2+ entry and IP3-dependent Ca2+ release.

Caffeine-induced Ca2+ oscillations in type I horizontal cells of the carp retina and the contribution of the store-operated Ca2+ entry pathway.

Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells.

Tumor necrosis factor modulates Ca2+ currents in cultured sympathetic neurons.

Soman reversibly decreases the duration of Ca2+ and Ba2+ action potentials in bullfrog sympathetic neurons.

The effects of soman on the electrical properties and excitability of bullfrog sympathetic ganglion neurones.

Store-operated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability.

Lithium Sensitive ORAI1 Expression, Store Operated Ca2+ Entry and Suicidal Death of Neurons in Chorea-Acanthocytosis.

Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation.

Store-operated Ca2+ entry supports contractile function in hearts of hibernators.

Trophic regulation of action potential in bullfrog sympathetic neurones.

Nanoscale patterning of STIM1 and Orai1 during store-operated Ca2+ entry.

Stretch-induced Ca2+ signalling in vascular smooth muscle cells depends on Ca2+ store segregation.

ADP evokes biphasic Ca2+ influx in fura-2-loaded human platelets. Evidence for Ca2+ entry regulated by the intracellular Ca2+ store.

Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration.

Effects of the antithrombitic agent PCA 4230 on agonist-induced Ca2+ entry and Ca2+ release in human platelets.

Sigma1 receptors inhibit store-operated Ca2+ entry by attenuating coupling of STIM1 to Orai1.