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Journal of Physiology (1992), 458, pp. 119-137 With 10 figures Printed in Great Britain
CONTRIBUTION OF Ca2`-INDUCED Ca2+ RELEASE TO THE [Ca2+1i TRANSIENTS IN MYOCYTES FROM GUINEA-PIG URINARY BLADDER BY V. YA. GANITKEVICH AND G. ISENBERG From the Department of Physiology, University of Cologne, Robert-Koch strafle 39, D-5000 Koln-41, Germany
(Received 3 January 1992) SUMMARY
1. Smooth muscle cells from guinea-pig urinary bladder were studied at an extracellular Ca2+ concentration ([Ca2+]0) of 3'6 mm and 36 'C. Fluorescence of Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transients) induced by step membrane depolarizations. 2. During a 6 s depolarization step from -60 to 0 mV [Ca2+]i increased from a resting 118 + 22 nm to 1150 + 336 nm and decayed to a sustained level of 295 + 62 nM. The experiments were designed to evaluate the contribution of the release of intracellularly stored Ca2+ to components of the depolarization-induced [Ca2+]i transient, i.e. 'phasic', which decayed during a maintained depolarization step, and 'tonic' which constituted the sustained elevation of [Ca2+]i above resting level. 3. A short (1 s) application of 10 mm caffeine mimicked the phasic component. After wash-out of caffeine, the subsequent depolarization induced a [Ca2+]i transient with reduced peak, the degree of suppression depending on the interval between wash-out of caffeine and depolarization. The phasic component of the depolarization and the caffeine-induced [Ca2+]i transients were not additive but saturative. 4. The phasic component was largely abolished in the continuous presence of 10 mm caffeine. It was also abolished by a 10 min cell dialysis of 10 /tM ryanodine from the pipette solution and was strongly reduced by dialysis of 5 /tM thapsigargin. Changes of the tonic component of the depolarization-induced [Ca2+]i transient were much less pronounced with all three interventions. 5. The tonic component of the depolarization-induced [Ca2+]i transient was increased when [Ca2+]. was elevated briefly before a depolarization close to 0 mV, whereas the phasic component was not significantly changed. Similarly, brief application of 1 /tM Bay K 8644 increased the tonic component several-fold without modifying significantly the phasic component. 6. It is concluded that depolarization-induced influx of Ca21 through L-type Ca2+ channels induces the release of Ca2+ from intracellular caffeine-sensitive stores which constitutes the major part of the phasic component. Ca2+ release superimposes on the effects of Ca2+ influx through L-type Ca2+ channels, the non-inactivating part of which constitutes the tonic component of the [Ca2+]i transient. Since the two processes interact, a dissection by simple subtraction is not possible. MS 1011 5
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V. YA. GANITKEVICH AND G. ISENBERG INTRODUCTION
Smooth muscle cells respond to the depolarization of their membrane with a contraction which is the result of an increase in cytosolic concentration of Ca2+ ions The details of such a depolarization-induced increase in [Ca2+]i can be studied when the voltage clamp of isolated cells is combined with microspectrofluometry (Becker, Singer, Walsh & Fay, 1989; Ganitkevich & Isenberg, 1991; Pacaud & Bolton, 1991; Schneider, Hopp & Isenberg, 1991). For smooth muscle cells, there is general agreement that depolarization can increase [Ca2+]i by activating Ca2+ influx through L-type Ca21 channels ('Ca). Whether this Ca2+ influx induces concomitant Ca2+ release from intracellular stores, however, is a question of debate (Jino, 1989, 1990a). This uncertainty contrasts with cardiac muscle, where the concept of Ca2+-induced release of Ca2+ (CIRC) from the sarcoplasic reticulum (SR) is well established, in experiments with skinned preparations (Fabiato, 1983, 1985) as well as with intact cells studied with microspectrofluometry under voltage clamp (Beuckelman & Wier, 1988; Wier, 1990). In smooth muscle, results from skinned preparations seemed to suggest that CIRC is unlikely to occur (lino, 1989, 1990a), and up to now microspectrofluometry under voltage clamp was used only to evaluate the importance of Ca21 influx in incrementing of [Ca2+]i (Becker et al. 1989; Ganitkevich & Isenberg, 1991) rather than in triggering CIRC. In the presence of CIRC the [Ca2+]i signal evoked by depolarization should contain two components, one due to Ca21 influx and another due to the release of intracellularly stored Ca21 via a CIRC mechanism. In the myocytes from the urinary bladder, Ca2+ influx through Na+-Ca2+ exchange is of minor importance and Ca2+ influx is mostly through L-type Ca21 channels (Ganitkevich & Isenberg, 1991). In the present study the contribution of Ca2+ influx was evaluated by applying fast changes in or by adding the Ca21 channel agonist Bay K 8644. However, changes in are ICa expected to also modify the trigger for CIRC and the extent of filling of the store from which the Ca21 is released. Therefore, experiments were performed with putatively an optimal trigger (potential steps close to 0 mV) and with a short exposure to the altered [Ca2+]. using a technique of rapid solution change. The contribution of CIRC was studied with three pharmacological interventions. Caffeine was rapidly applied to the cell to activate the SR Ca2+-release channels (Rosseau & Meissner, 1989) followed by depletion of the SR of the releasable Ca2+ (Weber & Herz, 1968; Endo, 1977; Ganitkevich & Isenberg, 1992b). The plant alkaloid ryanodine should act in a similar way (Rosseau, Smith & Meissner, 1987; Jino, 1989; Hisayama, Takayanagi & Okamoto, 1990). Finally, the tumour promotor thapsigargin should inhibit the Ca2+-ATPase of the SR (Kwan, Takemura, Obie, Thastrup & Putney, 1990; Thastrup, Cullen, Dtrobak, Hynley & Dawson, 1990; Bian, Ghosh, Wang & Gill, 1991). The results of this study suggest that the changes of [Ca2+]i in response to the membrane depolarization in smooth muscle cells from the guinea-pig urinary bladder are derived from both Ca2+ influx and intracellular Ca2+ release. The phasic component of the depolarization-induced [Ca2+]i transient is mostly due to CIRC from an intracellular Ca2+ store which is sensitive to caffeine, ryanodine and to thapsigargin. The tonic component is mostly due to Ca2+ influx via L-type Ca21 channels. A more detailed analysis suggests that the processes of Ca2+
([Ca2`]j).
[Ca2+].
Ca2+ RELEASE IN SMOOTH MUSCLE CELLS 121 influx and CIRC are not independent but coupled through the changes in [Ca2+]i; therefore a simple subtraction procedure is invalid. Parts of this work have been presented to the Physiological Society (Ganitkevich & Isenberg, 1992a). METHODS
Adult guinea-pigs were killed by cervical dislocation, and then the urinary bladder was removed. The methods of cell isolation, recording of whole-cell membrane currents as well as the measurement and calibration of [Ca2+]i with Indo-1 were described recently (Ganitkevich & Isenberg, 1991). Details are presented in the preceding paper (Ganitkevich & Isenberg, 1992 b). At 36 C, the myocytes were continuously superfused with a physiological salt solution (PSS) composed of (mM): 150 NaCl, 3-6 CaCl2, 1-2 MgCl2, 5-4 KCl, 20 glucose, 5 HEPES, adjusted with NaOH to pH 7-4. When [Ca2+]. was increased to 10 mm, or when 10 mm caffeine were added, no corrections for osmolarity were made. The pipettes were filled with an intracellular solution containing (mM): 130 KCl, 2 Na2ATP, 3 MgCl2, 10 HEPES, 0-1 K51ndo-1, adjusted with NaOH to pH 7-2. For a fast application of caffeine (as well as solutions containing different [Ca2+]0 or Bay K 8644), a four-barrel glass pipette (0-1 mm each opening) was placed approximately 0-25 mm from the cell from which solutions were pressure-applied (for details, see Ganitkevich & Isenberg, 1992b). The results when appropriate are expressed as means + S.D. of the mean. Statistical significance (P < 0 05) was evaluated with a Student's paired t test. RESULTS
Phasic and tonic components of the depolarization-induced [Ca2+]i transient In this study, [Ca2+]i changes ([Ca2+]i transients) in single voltage-clamped smooth muscle cells were induced by a 6 s depolarization step from the holding potential -60 mV to 0 mV which resulted in Ca2+ influx with calcium currents (ICa). Figure 1 shows that the amplitude and time course of these [Ca2+]i transients were subjected to some variability with time. In the beginning of the experiment (Fig. 1, 2 min after start of Indo-1 loading, left traces) the depolarization caused a rapid rise in [Ca21]i from 135 nm to a peak of 1010 nm which was achieved within approximately 0 3 s. ICa during the potential step was rapidly masked by a Ca2+-activated K+ current (IK, Ca) that peaked to 3-5 nA and subsequently fell rapidly. During the continuous depolarization, [Ca2+]i fell to a minimal [Ca21]i of 350 nm. Then it increased again, the secondary increase being in parallel with the appearance of a transient IK, Ca. Finally, when repolarization to -60 mV deactivated ICa' [Ca2+]i rapidly returned to the resting 135 nm. Simultaneously, an inward tail current was recorded in some cells that was most likely a Ca2+-activated Cl- current. The resting [Ca2+]i 5 min after achievement of the whole-cell mode was slightly increased (150 nM, Fig. 1, middle traces). The [Ca2+]i transient rose to a peak of 950 nm at a lower rate, the peak being achieved 900 ms after start of the depolarizing step. The decay of the [Ca2+]i transient was slowed down after 5 min perfusion compared to the records obtained after 2 min. Concomitantly, the IK, Ca was reduced; it peaked now to 2-1 nA and transient IK, Ca during the depolarization step was no longer observed. The effects become more pronounced with time; the right traces in Fig. 1 were obtained 10 min after cell access. The peak of [Ca2+]i (900 nM) was lower and occurred later (1-2 s) compared to the initial trace (Fig. 1, compare right and left traces). Within another 10 min, peak of [Ca2+]i and of net inward current were further reduced to approximately 30% of the original values (not shown). 5-2
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The changes illustrated in Fig. 1 may have resulted partially from the Ca2+ buffering effect of the Indo-1 whose concentration is thought to increase with time and may attenuate the possible regenerative responses. In addition, there was a continuous slow 'run-down' process; the cell dialysis may wash out endogenous 2min
10min
5min
[Ca2+], (nM) 1010
0-28Ratio
41R0/470]
0.1 31
-2
t
_
_
0-05) while the tonic component was significantly changed from 180 + 54 nm to 314 + 83 nM (P = 0-01). When the [Ca2+]i transient was induced by depolarization steps to -30 mV, the situation was more complicated. At -30 mV the test potential is at the descending
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branch of the current-voltage relation and screening of surface charges due to changes in [Ca2+]i is important. At -30 mV, the change from 2 to 10 mm [Ca2l]. is expected to shift the activation curve to the right and to slow down the kinetics (Ganitkevich, Shuba & Smirnov, 1988). Figure 8B shows the [Ca2+]i transient in [Ca 2+] (nM)
A 0-29Ratio1
-950 600
410/470
1 nA
3s B
[Ca2+ k (nM) 775
0-26-
Ratio]
410/470
L130
0.13-
10-2 nA 3s
Fig. 9. Increase in the depolarization-induced [Ca2+]i transient produced by 1 ,UM Bay K 8644. Depolarization steps to 0 mV (A) and to -30 mV (B). Left traces in A and B represent control [Ca21]i transients, right traces 10 s after application of Bay K 8644. Lower traces in A and B represent membrane current.
response to a depolarization step to -30 mV (2 mm [Ca2+]0). [Ca21]i increased from resting 120 nm to 400 nm and decayed during depolarization to a value of 200 nm. After the change to 10 mm [Ca2+]., the [Ca2+]i transient peaked within 3 s to 520 nM, the late peak occurring together with a late peak of IK, ca. The sustained [Ca2+]i value of 240 nm was reached at the end of the depolarizing pulse. Thus, in this experiment at -30 mV the phasic and the tonic components of the [Ca2+]i transient were increased from 200 to 280 nm and from 80 to 120 nm when the [Ca2+]o was increased from 2 to 10 mM.
Modulation of the Ca2+ influx by Bay K 8644 In the present experiments during depolarizing steps the Ca2+ influx into the cells occurs through L-type Ca2+ channels. Since they are known to be sensitive to 'Ca2+ agonist' Bay K 8644, the effect of this drug was studied. When 1 ,UM Bay K 8644 was applied 10 s before the depolarization step to 0 mV, peak of net inward current was approximately doubled (Fig. 9A). As expected from the bigger Ca2+ influx, [Ca2+]i transient was increased in the presence of Bay K 8644. The phasic component was increased from 325 to 350 nm while the tonic component was increased from 100 to 450 nm. One can estimate that Ca2+ influx contributed 56% of the increment in
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[Caa21]i when Bay K 8644 was present but only 24 % before. In five cells studied with 1 ,UM Bay K 8644 this contribution was 22 + 6 % at 0 mV in control and 47 + 8 % in the presence of Bay K 8644. Bay K 8644 (1 /,M) did not increase the resting [Ca2+]i as long as no depolarizations were applied; however, the depolarization-induced [Ca21]i transients fell during Bay K 8644
[Cal+]i
(nM) 430
0-21Ratio 1 410/470 0-13 W;>
-126
1 nA|
0 mV, 20 s pulse -60 mV-
Fig. 10. Effect of the rapid application of 1 /SM Bay K 8644 on the [Ca2+]i transient (upper trace) during the 20 s depolarization step from -60 to 0 mV. Middle trace demonstrates membrane current and lower trace indicates the membrane potential. Note, Bay K 8644 markedly increased the tonic component of the [Ca2+]i transient.
repolarization very slowly (see Fig. 9A) and after several depolarizing pulses reached a resting [Ca2+]i value that was higher than in control. When the membrane was depolarized from -60 to -30 mV, the [Ca2+]i increased to 250 nm and slowly decayed approaching the value of 180 nm at the end of the pulse (Fig. 9B). After application of 1 UM Bay K 8644, the depolarization induced a [Ca2+]i transient in which the phasic component was increased from 70 to 425 nm and where the tonic component could be clearly distinguished, being 220 nm compared to 50 nM in control (Fig. 9B). On average, Bay K 8644 increased the tonic component at -30 mV from 83+ 13 nm to 242+39 nM (n = 4). Using the fast solution change Bay K 8644 could be applied during a 20 s long depolarization to 0 mV. Figure 10 shows the results of an experiment where Bay K 8644 was applied 3 s after the start of depolarization. Bay K 8644 increased [Ca2+]i within 6 s to a sustained value (430 nM) which was higher in this cell than the [Ca2+]i peak before the Bay K 8644 exposure. In parallel, the outward IK, ca was increased (Fig. 10). The equilibration period of 6 s can be attributed to the period necessary to increase the non-inactivating ICa as it was evaluated with a fast solution change (Hering, Beech, Bolton & Lim, 1988). The results are consistent with the measurements of ICa in these cells where the several-fold increase of non-inactivating ICa in the presence of Bay K 8644 was demonstrated (Imaizumi, Muraki, Takeda &
Watanabe, 1989).
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DISCUSSION
The results of this paper support the hypothesis that membrane depolarization increases [Ca2+]i in smooth muscle cells via both Ca2` influx from the extracellular space and Ca2" release from intracellular stores. The contribution of Ca2+ release was tested in the present study by interventions known to deplete the SR of releasable Ca2+, i.e. during the continuous exposure to 10 mm caffeine, to 10 /M ryanodine or to 5 tM thapsigargin. Thapsigargin was applied as an inhibitor of the SR Ca2+-ATPase (Missiaen et al. 1991). With the time of thapsigargin exposure the decay rate of the [Ca2+]i transients became progressively slower (Fig. 7A). This results suggests that the time course of the decay of the [Ca2+]i transient in the untreated cell is limited by the SR Ca2+ reuptake. After 10 min thapsigargin had suppressed the initial phasic component of the depolarization-induced [Ca2+]i transient, as expected when SR was depleted of releasable Ca2+. The phasic component was also suppressed by 10 /am intracellular ryanodine. Ryanodine binds specifically to the Ca2+-release channel ('ryanodine receptor') and locks it into a permanently open subconductive state. Under these conditions, the SR Ca2+-ATPase has to pump Ca2+ into a leaky SR. As a result, the SR cannot maintain the Ca2+ gradient and therefore its capability for Ca2+ release. Abolition of the phasic component of the [Ca2+]i transient (Figs 5 and 6) as well as the inability of the caffeine to induce Ca2+ release (Fig. 6) support this hypothesis. The third intervention for suppressing the phasic component used was a sustained application of caffeine. At a concentration of 10 mm, caffeine opens the Ca2+-release channels by sensitizing them for Ca2+ activation (Rousseau & Meissner, 1989; Sitsapesan & Williams, 1990); the concomitant Ca2+ release is indicated as a caffeineinduced [Ca2+]i transient (Fig. 2). In the continuous presence of 10 mm caffeine the SR is depleted of releasable Ca2+ (Ganitkevich & Isenberg, 1992 b), therefore the phasic component of the [Ca2+]i transient was abolished (Fig. 4). In addition the results demonstrated that the caffeine- and depolarization-induced [Ca2+]i transients were not additive but saturative in triggering the increase in [Ca2+]j, as expected when both procedures release Ca2+ from the same store. Each of the three interventions has its shortcomings. In the case of thapsigargin and ryanodine, the effects required several minutes to develop and may be, thus, contaminated by a 'run-down' of the [Ca2+]i transients. However, the phasic component of the [Ca2+]i transient was not abolished in control cells within 10 min (Fig. 1), as it was in ryanodine- and thapsigargin-treated cells (Figs 5, 6 and 7). In addition, there is evidence that only some of the SR Ca2+ stores are sensitive to thapsigargin (Missiaen et al. 1991). Vice versa, ryanodine and caffeine interact only with the ryanodine, but not with the IP3, receptors, e.g. part of the SR may still contain some releasable Ca2+ (Jino, 1990b; but see Pacaud & Bolton, 1991). For all three interventions, the suppression of the phasic component was very similar. This similarity strongly supports the hypothesis that depolarization triggers the release from that part of the SR that might be called the 'caffeine-sensitive Ca2+ store' (Iino, 1989; Ganitkevich & Isenberg, 1992b). In analogy to the Ca2+ release from cardiac SR (Fabiato, 1983, 1985) we suggest that the underlying mechanism is the
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'Ca2+-induced release of Ca2+' (CIRC) with Ca2+ influx through Ca2+ channels as release trigger (see below). The Ca2+ release contributes a transient increase and decay of the [Ca2+]i change following the membrane depolarization which is called 'phasic' in the present work. It should be mentioned also, however, that Ca2+ influx generates a time-dependent [Ca2+]i change, as long as ICa activates and inactivates. Thus, the [Ca2+]i transients obtained with suppressed CIRC were not time independent but rose to a peak and slowly decayed (Figs 4, 5, 6 and 7). After several seconds, however, when ICa inactivation had become steady, Ca2+ influx occurred at a constant rate, and the sustained [Ca2+]j became time independent at a level where Ca2+ influx was balanced by Ca2+ extrusion. Although the results are clear in a qualitative way, they do not permit a quantitative dissection of the [Ca2+]i transient into a component due to Ca2+ release and a component due to Ca2+ influx. The difficulty arises because the [Ca2+]i transient also contains components of the redistribution of Ca2+; a simple subtraction of the [Ca2+]i transient recorded in the presence of the thapsigargin from the control recording would contain the contributions of suppressed release, suppressed reuptake plus those of stimulated Ca2+ extrusion. Since the components are not independent of each other a simple subtraction is not possible. For example, thapsigargin (as well as ryanodine) increased the 'resting' [Ca2+]i and due to Ca2+-induced inactivation of L-type Ca2+ channels the amplitude of ICa was diminished. In the case of caffeine, direct inhibition of the Ca2+ channels has been reported (Hughes, Hering & Bolton, 1990). Thus, when Ca2+ influx was reduced below control level by the above interventions, the contribution of control Ca2+ influx to the depolarization-induced [Ca2+]i transient is expected to be underestimated. The results of the present study may suggest that at 3-6 mm [Ca2+]0 and 36 °C with membrane potential steps to 0 mV approximately 70 % of the increment in [Ca2+]i is due to CIRC and only 30% due to Ca2+ influx. The relative importance of the two processes, however, depends on a variety of factors including the time after beginning of the depolarization. This value can increase to about 50% when ICa is increased by elevation in [Ca2+]o (Fig. 8) or by addition of a Ca2+ agonist (Figs 9 and 10). The relative importance of CIRC can decrease to values below 50% with submaximal activation, i.e. during graded responses, when the trigger of Ca2+ release is reduced. An example was given for depolarizing pulses to -30 mV, in the presence and absence of Bay K 8644 (Fig. 9B). The trigger for CIRC also diminishes when the duration of the depolarizing step was shortened to less than 300 ms (Ganitkevich & Isenberg, 1991). Thus, the question of the contribution of CIRC and Ca2+ influx in other experimental conditions, like during one or several action potentials, has to be evaluated in future. Compared to [Ca2+]i transients in cardiac preparations, where the role of CIRC is well established (Beuckelmann & Wier, 1988; Wendt-Galitelli & Isenberg, 1991), in smooth muscle cells under study the depolarization-induced [Ca2+]i transient peaked within approximately 300 ms (or even more) while in a cardiac specimen within about 20 ms. A recent study of reconstituted ryanodine receptors indicated that the release channel from vascular muscle has a similar [Ca2+]i sensitivity as the release channel from skeletal or cardiac SR (Herrmann-Frank, Darling & Meissner, 1991). If
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it is not the channel protein per se, then the ultrastructural organization of the SR could be responsible for the difference. For example, it is possible that some parts of SR in smooth muscle cells are located at different distances from the surface membrane than others. Activation of CIRC there is expected to occur with some diffusional delay; diffusion could be also hindered by the membranes of peripheral SR. This possibility is indirectly supported by the result that IK Ca' which is activated by Ca2+ ions close to the inner side of the surface membrane, peaked and fell faster than the global [Ca2+]i as it is monitored by the Indo- 1 (see Figs 1, 3A and 4A). The dye Indo-1 competes with native intracellular Ca2+ binding sites, e.g. of proteins. The concentration of the dye (100/tM) was low in comparison with the endogenous Ca2+ buffer, estimated to be > 1 mm (Becker, Walsh, Singer & Fay, 1988). Interventions which resulted in the increase of the resting [Ca2+]i are expected to saturate the endogenous Ca2+ buffer to a higher degree. Competition of these sites with the Indo-1 would be less, and the dye signal may rise at higher rate. Thus, the 'square-like' [Ca2]i transient that was recorded in the presence of ryanodine from a resting [Ca2+]i of 200 nm should not be extrapolated to the control. A similar argument holds true for the contribution of peak and sustained ICa to the [Ca2+]i change. At the beginning of the depolarization [Ca2+]i is low and a large amount of inflowing Ca2+ should be buffered. During the sustained level [Ca2+]i is elevated to 300-500 nm, part of the buffer should be saturated and the relative small variation Of ICa through non-inactivating L-type channels (about 20 pA, Imaizumi et al. 1989) can modulate [Ca2+]i more effectively. Thus, when the degree of Ca2+ binding is different, the time course of the [Ca2+]i should not be subtracted. We are grateful to Professor D. A. Eisner for his comments on the manuscript.
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BECKER, P. L., SINGER, J. J., WALSH, J. V. JR & FAY, F. S. (1989). Regulation of calcium concentration in voltage clamped smooth muscle cells. Science 244, 211-214. BECKER, P. L., WALSH, J. V., SINGER, J. J. & FAY, F. S. (1988). Calcium buffering capacity, calcium currents, and [Ca2+] changes in voltage clamped, Fura-2 loaded single smooth muscle cells. Biophysical Journal 53, 595 a. BEUCKELMANN, D. J. & WIER, W. G. (1988). Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells. Journal of Physiology 405, 233-255. BIAN, J., GHOSH, T. K., WANG, J. C. & GILL, D. L. (1991). Identification of intracellular calcium pools. Selective modification by thapsigargin. Journal of Biological Chemistry 266, 8801-8806. ENDO, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiological Reviews 57, 71-108. FABIATO, A. (1983). Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. American Journal of Physiology 245, C1-14. FABIATO, A. (1985). Time and calcium dependence of activation and inactivation of calciuminduced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell. Journal of General Physiology 85, 247-289. GANITKEVICH, V. YA. & ISENBERG, G. (1991). Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder. Journal of Physiology 435, 187-205.
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GANITKEVICH, V. YA. & ISENBERG, G. (1992a). Contribution of Ca2+-induced Ca2' release to the depolarization-induced Ca2+ transients in smooth muscle cells from the guinea-pig urinary bladder. Journal of Physiology 446, 140P. GANITKEVICH, V. YA. & ISENBERG, G. (1992b). Caffeine-induced release and reuptake of Ca2+ by Ca2" stores in myocytes from the guinea-pig urinary bladder. Journal of Physiology 458, 99-117. GANITKEVICH, V. YA., SHUBA, M. F. & SMIRNOV, S. V. (1987). Calcium-dependent inactivation of potential-dependent calcium inward current in an isolated guinea-pig smooth muscle cell. Journal of Physiology 392, 431-449. GANITKEVICH, V. YA., SHUBA, M. F. & SMIRNOV, S. V. (1988). Saturation of calcium channels in single isolated smooth muscle cells of guinea-pig taenia caeci. Journal of Physiology 399, 419-436. HERING, S., BEECH, D. J., BOLTON, T. B. & LIM, S. P. (1988). Action of nifedipine or Bay K 8644 is dependent on calcium channel state in single smooth muscle cells from rabbit ear artery.
Pfiiigers Archiv 411, 590-592. HERRMANN-FRANK, A., DARLING, E. & MEISSNER, G. (1991). Functional characterization of the C(a2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum. Pftilgers Archiv 418, 353-359. HISAYAMA, T., TAKAYANAGI, I. & OKAMOTO, Y. (1990). Ryanodine reveals multiple contractile and relaxant mechanisms in vascular smooth muscle: simultaneous measurements of mechanical activity and of cytoplasmic free Ca2+ level with Fura-2. British Journal of Pharmacology 100, 677-684. HUGHES, A. D., HERING, S. & BOLTON, T. B. (1990). The action of caffeine on inward barium current through voltage-dependent calcium channels in single rabbit ear artery cells. Pfluigers Archiv 416, 462-466. IINO, M. (1989). Calcium-induced calcium release mechanism in guinea pig taenia caeci. Journal of General Physiology 94, 363-383. IINO, M. (1990a). Biphasic Ca2+ dependence of inositol 1,4,5-triphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. Journal of General Physiology 95, 1103-1122. IINO, M. (1990b). Calcium release mechanisms in smooth muscle. Japanese Journal of Pharmacology 54, 345-354. IMAIZUMI, Y., MURAKI, K., TAKEDA, M. & WATANABE, M. (1989). Measurement and simulation of noninactivating Ca current in smooth muscle cells. American Journal of Physiology 256, C880-885. KANMURA, Y., MISSIAEN, L., RAEYMAEKERS, L. & CASTEELS, R. (1988). Ryanodine reduces the amount of calcium in intracellular stores of smooth-muscle cells of the rabbit ear artery. Pfiilgers Archiv 413, 153-159. KOMORI, S. & BOLTON, T. B. (1991). Inositol triphosphate releases stored calcium to block voltagedependent calcium channels in single smooth muscle cells. Pfluigers Archiv 418, 437-441. KWAN, C. Y., TAKEMURA, H., OBIE, J. F., THASTRUP, 0. & PUTNEY, J. W. JR (1990). Effects of MeCh, thapsigargin, and La3+ on plasmalemmal and intracellular Ca2+ transport in lacrimal acinar cells. American Journal of Physiology 258, C1006-1015. MISSIAEN, L., WUYTACK, F., RAEYMAEKERS, L., DE SMEDT, H., DROOGMANS, G., DECLERCK,I. & CASTEELS, R. (1991). Ca2' extrusion across plasma membrane and Ca2+ uptake by intracellular stores. Pharmaceutical Therapeutics 50, 191-232. O'NEILL, S. C., DONOSO, P. & EISNER, D. A. (1990). The role of [Ca2+]i and [Ca2+] sensitization in the caffeine contracture of rat myocytes: measurement of [Ca2+]i and [caffeine]j. Journal of Physiology 425, 55-70. PACAUD, P. & BOLTON, T. B. (1991). Relation between muscarinic receptor cationic current and internal calcium in guinea-pig jejunal smooth muscle cells. Journal of Physiology 441, 477-499. ROUSSEAU, E. & MEISSNER, G. (1989). Single cardiac sarcoplasmic reticulum Ca2+-release channels: activation by caffeine. American Journal of Physiology 256, H328-333. ROUSSEAU, E., SMITH, J. S. & MEISSNER, G. (1987). Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. American Journal of Physiology 253, C364-368. SCHNEIDER, P., Hopp, H. H. & ISENBERG, G. (1991). Ca2+ influx through ATP-gated channels increments [Ca2+]i and inactivatesICa in myocytes from guinea-pig urinary bladder. Journal of Physiology 440, 479-496. SITSAPESAN, R. & WILLIAMS, A. J. (1990). Mechanisms of caffeine activation of single calciumrelease channels of sheep cardiac sarcoplasmic reticulum. Journal of Physiology 423, 425-439.
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THASTRUP, O., CULLEN, P. J., DTROBAK, B. K., HYNLEY, M. R. & DAWSON, A. P. (1990). Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proceedings of the National Academy of Sciences of the USA 87, 2466-2470. WEBER, A. M. & HERZ, R. (1968). The relationship between caffeine contracture of intact muscle and the effect of caffeine on isolated reticulum. Journal of General Physiology 52, 750-759. WENDT-GALITELLI, M. F. & ISENBERG, G. (1991). Total and free myoplasmic calcium during a contraction cycle: X-ray microanalysis in guinea-pig ventricular myocytes. Journal ofPhysiology 435, 349-372. WIER, W. G. (1990). Cytoplasmic [Ca2+] in mammalian ventricle: dynamic control by cellular processes. Annual Review of Physiology 52, 467-485.
Journal of Physiology (1992), 458, pp. 119-137 With 10 figures Printed in Great Britain
CONTRIBUTION OF Ca2`-INDUCED Ca2+ RELEASE TO THE [Ca2+1i TRANSIENTS IN MYOCYTES FROM GUINEA-PIG URINARY BLADDER BY V. YA. GANITKEVICH AND G. ISENBERG From the Department of Physiology, University of Cologne, Robert-Koch strafle 39, D-5000 Koln-41, Germany
(Received 3 January 1992) SUMMARY
1. Smooth muscle cells from guinea-pig urinary bladder were studied at an extracellular Ca2+ concentration ([Ca2+]0) of 3'6 mm and 36 'C. Fluorescence of Indo-1 was used to monitor the cytosolic calcium concentration ([Ca2+]i) and its changes ([Ca2+]i transients) induced by step membrane depolarizations. 2. During a 6 s depolarization step from -60 to 0 mV [Ca2+]i increased from a resting 118 + 22 nm to 1150 + 336 nm and decayed to a sustained level of 295 + 62 nM. The experiments were designed to evaluate the contribution of the release of intracellularly stored Ca2+ to components of the depolarization-induced [Ca2+]i transient, i.e. 'phasic', which decayed during a maintained depolarization step, and 'tonic' which constituted the sustained elevation of [Ca2+]i above resting level. 3. A short (1 s) application of 10 mm caffeine mimicked the phasic component. After wash-out of caffeine, the subsequent depolarization induced a [Ca2+]i transient with reduced peak, the degree of suppression depending on the interval between wash-out of caffeine and depolarization. The phasic component of the depolarization and the caffeine-induced [Ca2+]i transients were not additive but saturative. 4. The phasic component was largely abolished in the continuous presence of 10 mm caffeine. It was also abolished by a 10 min cell dialysis of 10 /tM ryanodine from the pipette solution and was strongly reduced by dialysis of 5 /tM thapsigargin. Changes of the tonic component of the depolarization-induced [Ca2+]i transient were much less pronounced with all three interventions. 5. The tonic component of the depolarization-induced [Ca2+]i transient was increased when [Ca2+]. was elevated briefly before a depolarization close to 0 mV, whereas the phasic component was not significantly changed. Similarly, brief application of 1 /tM Bay K 8644 increased the tonic component several-fold without modifying significantly the phasic component. 6. It is concluded that depolarization-induced influx of Ca21 through L-type Ca2+ channels induces the release of Ca2+ from intracellular caffeine-sensitive stores which constitutes the major part of the phasic component. Ca2+ release superimposes on the effects of Ca2+ influx through L-type Ca2+ channels, the non-inactivating part of which constitutes the tonic component of the [Ca2+]i transient. Since the two processes interact, a dissection by simple subtraction is not possible. MS 1011 5
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V. YA. GANITKEVICH AND G. ISENBERG INTRODUCTION
Smooth muscle cells respond to the depolarization of their membrane with a contraction which is the result of an increase in cytosolic concentration of Ca2+ ions The details of such a depolarization-induced increase in [Ca2+]i can be studied when the voltage clamp of isolated cells is combined with microspectrofluometry (Becker, Singer, Walsh & Fay, 1989; Ganitkevich & Isenberg, 1991; Pacaud & Bolton, 1991; Schneider, Hopp & Isenberg, 1991). For smooth muscle cells, there is general agreement that depolarization can increase [Ca2+]i by activating Ca2+ influx through L-type Ca21 channels ('Ca). Whether this Ca2+ influx induces concomitant Ca2+ release from intracellular stores, however, is a question of debate (Jino, 1989, 1990a). This uncertainty contrasts with cardiac muscle, where the concept of Ca2+-induced release of Ca2+ (CIRC) from the sarcoplasic reticulum (SR) is well established, in experiments with skinned preparations (Fabiato, 1983, 1985) as well as with intact cells studied with microspectrofluometry under voltage clamp (Beuckelman & Wier, 1988; Wier, 1990). In smooth muscle, results from skinned preparations seemed to suggest that CIRC is unlikely to occur (lino, 1989, 1990a), and up to now microspectrofluometry under voltage clamp was used only to evaluate the importance of Ca21 influx in incrementing of [Ca2+]i (Becker et al. 1989; Ganitkevich & Isenberg, 1991) rather than in triggering CIRC. In the presence of CIRC the [Ca2+]i signal evoked by depolarization should contain two components, one due to Ca21 influx and another due to the release of intracellularly stored Ca21 via a CIRC mechanism. In the myocytes from the urinary bladder, Ca2+ influx through Na+-Ca2+ exchange is of minor importance and Ca2+ influx is mostly through L-type Ca21 channels (Ganitkevich & Isenberg, 1991). In the present study the contribution of Ca2+ influx was evaluated by applying fast changes in or by adding the Ca21 channel agonist Bay K 8644. However, changes in are ICa expected to also modify the trigger for CIRC and the extent of filling of the store from which the Ca21 is released. Therefore, experiments were performed with putatively an optimal trigger (potential steps close to 0 mV) and with a short exposure to the altered [Ca2+]. using a technique of rapid solution change. The contribution of CIRC was studied with three pharmacological interventions. Caffeine was rapidly applied to the cell to activate the SR Ca2+-release channels (Rosseau & Meissner, 1989) followed by depletion of the SR of the releasable Ca2+ (Weber & Herz, 1968; Endo, 1977; Ganitkevich & Isenberg, 1992b). The plant alkaloid ryanodine should act in a similar way (Rosseau, Smith & Meissner, 1987; Jino, 1989; Hisayama, Takayanagi & Okamoto, 1990). Finally, the tumour promotor thapsigargin should inhibit the Ca2+-ATPase of the SR (Kwan, Takemura, Obie, Thastrup & Putney, 1990; Thastrup, Cullen, Dtrobak, Hynley & Dawson, 1990; Bian, Ghosh, Wang & Gill, 1991). The results of this study suggest that the changes of [Ca2+]i in response to the membrane depolarization in smooth muscle cells from the guinea-pig urinary bladder are derived from both Ca2+ influx and intracellular Ca2+ release. The phasic component of the depolarization-induced [Ca2+]i transient is mostly due to CIRC from an intracellular Ca2+ store which is sensitive to caffeine, ryanodine and to thapsigargin. The tonic component is mostly due to Ca2+ influx via L-type Ca21 channels. A more detailed analysis suggests that the processes of Ca2+
([Ca2`]j).
[Ca2+].
Ca2+ RELEASE IN SMOOTH MUSCLE CELLS 121 influx and CIRC are not independent but coupled through the changes in [Ca2+]i; therefore a simple subtraction procedure is invalid. Parts of this work have been presented to the Physiological Society (Ganitkevich & Isenberg, 1992a). METHODS
Adult guinea-pigs were killed by cervical dislocation, and then the urinary bladder was removed. The methods of cell isolation, recording of whole-cell membrane currents as well as the measurement and calibration of [Ca2+]i with Indo-1 were described recently (Ganitkevich & Isenberg, 1991). Details are presented in the preceding paper (Ganitkevich & Isenberg, 1992 b). At 36 C, the myocytes were continuously superfused with a physiological salt solution (PSS) composed of (mM): 150 NaCl, 3-6 CaCl2, 1-2 MgCl2, 5-4 KCl, 20 glucose, 5 HEPES, adjusted with NaOH to pH 7-4. When [Ca2+]. was increased to 10 mm, or when 10 mm caffeine were added, no corrections for osmolarity were made. The pipettes were filled with an intracellular solution containing (mM): 130 KCl, 2 Na2ATP, 3 MgCl2, 10 HEPES, 0-1 K51ndo-1, adjusted with NaOH to pH 7-2. For a fast application of caffeine (as well as solutions containing different [Ca2+]0 or Bay K 8644), a four-barrel glass pipette (0-1 mm each opening) was placed approximately 0-25 mm from the cell from which solutions were pressure-applied (for details, see Ganitkevich & Isenberg, 1992b). The results when appropriate are expressed as means + S.D. of the mean. Statistical significance (P < 0 05) was evaluated with a Student's paired t test. RESULTS
Phasic and tonic components of the depolarization-induced [Ca2+]i transient In this study, [Ca2+]i changes ([Ca2+]i transients) in single voltage-clamped smooth muscle cells were induced by a 6 s depolarization step from the holding potential -60 mV to 0 mV which resulted in Ca2+ influx with calcium currents (ICa). Figure 1 shows that the amplitude and time course of these [Ca2+]i transients were subjected to some variability with time. In the beginning of the experiment (Fig. 1, 2 min after start of Indo-1 loading, left traces) the depolarization caused a rapid rise in [Ca21]i from 135 nm to a peak of 1010 nm which was achieved within approximately 0 3 s. ICa during the potential step was rapidly masked by a Ca2+-activated K+ current (IK, Ca) that peaked to 3-5 nA and subsequently fell rapidly. During the continuous depolarization, [Ca2+]i fell to a minimal [Ca21]i of 350 nm. Then it increased again, the secondary increase being in parallel with the appearance of a transient IK, Ca. Finally, when repolarization to -60 mV deactivated ICa' [Ca2+]i rapidly returned to the resting 135 nm. Simultaneously, an inward tail current was recorded in some cells that was most likely a Ca2+-activated Cl- current. The resting [Ca2+]i 5 min after achievement of the whole-cell mode was slightly increased (150 nM, Fig. 1, middle traces). The [Ca2+]i transient rose to a peak of 950 nm at a lower rate, the peak being achieved 900 ms after start of the depolarizing step. The decay of the [Ca2+]i transient was slowed down after 5 min perfusion compared to the records obtained after 2 min. Concomitantly, the IK, Ca was reduced; it peaked now to 2-1 nA and transient IK, Ca during the depolarization step was no longer observed. The effects become more pronounced with time; the right traces in Fig. 1 were obtained 10 min after cell access. The peak of [Ca2+]i (900 nM) was lower and occurred later (1-2 s) compared to the initial trace (Fig. 1, compare right and left traces). Within another 10 min, peak of [Ca2+]i and of net inward current were further reduced to approximately 30% of the original values (not shown). 5-2
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The changes illustrated in Fig. 1 may have resulted partially from the Ca2+ buffering effect of the Indo-1 whose concentration is thought to increase with time and may attenuate the possible regenerative responses. In addition, there was a continuous slow 'run-down' process; the cell dialysis may wash out endogenous 2min
10min
5min
[Ca2+], (nM) 1010
0-28Ratio
41R0/470]
0.1 31
-2
t
_
_
0-05) while the tonic component was significantly changed from 180 + 54 nm to 314 + 83 nM (P = 0-01). When the [Ca2+]i transient was induced by depolarization steps to -30 mV, the situation was more complicated. At -30 mV the test potential is at the descending
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branch of the current-voltage relation and screening of surface charges due to changes in [Ca2+]i is important. At -30 mV, the change from 2 to 10 mm [Ca2l]. is expected to shift the activation curve to the right and to slow down the kinetics (Ganitkevich, Shuba & Smirnov, 1988). Figure 8B shows the [Ca2+]i transient in [Ca 2+] (nM)
A 0-29Ratio1
-950 600
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3s B
[Ca2+ k (nM) 775
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Ratio]
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Fig. 9. Increase in the depolarization-induced [Ca2+]i transient produced by 1 ,UM Bay K 8644. Depolarization steps to 0 mV (A) and to -30 mV (B). Left traces in A and B represent control [Ca21]i transients, right traces 10 s after application of Bay K 8644. Lower traces in A and B represent membrane current.
response to a depolarization step to -30 mV (2 mm [Ca2+]0). [Ca21]i increased from resting 120 nm to 400 nm and decayed during depolarization to a value of 200 nm. After the change to 10 mm [Ca2+]., the [Ca2+]i transient peaked within 3 s to 520 nM, the late peak occurring together with a late peak of IK, ca. The sustained [Ca2+]i value of 240 nm was reached at the end of the depolarizing pulse. Thus, in this experiment at -30 mV the phasic and the tonic components of the [Ca2+]i transient were increased from 200 to 280 nm and from 80 to 120 nm when the [Ca2+]o was increased from 2 to 10 mM.
Modulation of the Ca2+ influx by Bay K 8644 In the present experiments during depolarizing steps the Ca2+ influx into the cells occurs through L-type Ca2+ channels. Since they are known to be sensitive to 'Ca2+ agonist' Bay K 8644, the effect of this drug was studied. When 1 ,UM Bay K 8644 was applied 10 s before the depolarization step to 0 mV, peak of net inward current was approximately doubled (Fig. 9A). As expected from the bigger Ca2+ influx, [Ca2+]i transient was increased in the presence of Bay K 8644. The phasic component was increased from 325 to 350 nm while the tonic component was increased from 100 to 450 nm. One can estimate that Ca2+ influx contributed 56% of the increment in
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[Caa21]i when Bay K 8644 was present but only 24 % before. In five cells studied with 1 ,UM Bay K 8644 this contribution was 22 + 6 % at 0 mV in control and 47 + 8 % in the presence of Bay K 8644. Bay K 8644 (1 /,M) did not increase the resting [Ca2+]i as long as no depolarizations were applied; however, the depolarization-induced [Ca21]i transients fell during Bay K 8644
[Cal+]i
(nM) 430
0-21Ratio 1 410/470 0-13 W;>
-126
1 nA|
0 mV, 20 s pulse -60 mV-
Fig. 10. Effect of the rapid application of 1 /SM Bay K 8644 on the [Ca2+]i transient (upper trace) during the 20 s depolarization step from -60 to 0 mV. Middle trace demonstrates membrane current and lower trace indicates the membrane potential. Note, Bay K 8644 markedly increased the tonic component of the [Ca2+]i transient.
repolarization very slowly (see Fig. 9A) and after several depolarizing pulses reached a resting [Ca2+]i value that was higher than in control. When the membrane was depolarized from -60 to -30 mV, the [Ca2+]i increased to 250 nm and slowly decayed approaching the value of 180 nm at the end of the pulse (Fig. 9B). After application of 1 UM Bay K 8644, the depolarization induced a [Ca2+]i transient in which the phasic component was increased from 70 to 425 nm and where the tonic component could be clearly distinguished, being 220 nm compared to 50 nM in control (Fig. 9B). On average, Bay K 8644 increased the tonic component at -30 mV from 83+ 13 nm to 242+39 nM (n = 4). Using the fast solution change Bay K 8644 could be applied during a 20 s long depolarization to 0 mV. Figure 10 shows the results of an experiment where Bay K 8644 was applied 3 s after the start of depolarization. Bay K 8644 increased [Ca2+]i within 6 s to a sustained value (430 nM) which was higher in this cell than the [Ca2+]i peak before the Bay K 8644 exposure. In parallel, the outward IK, ca was increased (Fig. 10). The equilibration period of 6 s can be attributed to the period necessary to increase the non-inactivating ICa as it was evaluated with a fast solution change (Hering, Beech, Bolton & Lim, 1988). The results are consistent with the measurements of ICa in these cells where the several-fold increase of non-inactivating ICa in the presence of Bay K 8644 was demonstrated (Imaizumi, Muraki, Takeda &
Watanabe, 1989).
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DISCUSSION
The results of this paper support the hypothesis that membrane depolarization increases [Ca2+]i in smooth muscle cells via both Ca2` influx from the extracellular space and Ca2" release from intracellular stores. The contribution of Ca2+ release was tested in the present study by interventions known to deplete the SR of releasable Ca2+, i.e. during the continuous exposure to 10 mm caffeine, to 10 /M ryanodine or to 5 tM thapsigargin. Thapsigargin was applied as an inhibitor of the SR Ca2+-ATPase (Missiaen et al. 1991). With the time of thapsigargin exposure the decay rate of the [Ca2+]i transients became progressively slower (Fig. 7A). This results suggests that the time course of the decay of the [Ca2+]i transient in the untreated cell is limited by the SR Ca2+ reuptake. After 10 min thapsigargin had suppressed the initial phasic component of the depolarization-induced [Ca2+]i transient, as expected when SR was depleted of releasable Ca2+. The phasic component was also suppressed by 10 /am intracellular ryanodine. Ryanodine binds specifically to the Ca2+-release channel ('ryanodine receptor') and locks it into a permanently open subconductive state. Under these conditions, the SR Ca2+-ATPase has to pump Ca2+ into a leaky SR. As a result, the SR cannot maintain the Ca2+ gradient and therefore its capability for Ca2+ release. Abolition of the phasic component of the [Ca2+]i transient (Figs 5 and 6) as well as the inability of the caffeine to induce Ca2+ release (Fig. 6) support this hypothesis. The third intervention for suppressing the phasic component used was a sustained application of caffeine. At a concentration of 10 mm, caffeine opens the Ca2+-release channels by sensitizing them for Ca2+ activation (Rousseau & Meissner, 1989; Sitsapesan & Williams, 1990); the concomitant Ca2+ release is indicated as a caffeineinduced [Ca2+]i transient (Fig. 2). In the continuous presence of 10 mm caffeine the SR is depleted of releasable Ca2+ (Ganitkevich & Isenberg, 1992 b), therefore the phasic component of the [Ca2+]i transient was abolished (Fig. 4). In addition the results demonstrated that the caffeine- and depolarization-induced [Ca2+]i transients were not additive but saturative in triggering the increase in [Ca2+]j, as expected when both procedures release Ca2+ from the same store. Each of the three interventions has its shortcomings. In the case of thapsigargin and ryanodine, the effects required several minutes to develop and may be, thus, contaminated by a 'run-down' of the [Ca2+]i transients. However, the phasic component of the [Ca2+]i transient was not abolished in control cells within 10 min (Fig. 1), as it was in ryanodine- and thapsigargin-treated cells (Figs 5, 6 and 7). In addition, there is evidence that only some of the SR Ca2+ stores are sensitive to thapsigargin (Missiaen et al. 1991). Vice versa, ryanodine and caffeine interact only with the ryanodine, but not with the IP3, receptors, e.g. part of the SR may still contain some releasable Ca2+ (Jino, 1990b; but see Pacaud & Bolton, 1991). For all three interventions, the suppression of the phasic component was very similar. This similarity strongly supports the hypothesis that depolarization triggers the release from that part of the SR that might be called the 'caffeine-sensitive Ca2+ store' (Iino, 1989; Ganitkevich & Isenberg, 1992b). In analogy to the Ca2+ release from cardiac SR (Fabiato, 1983, 1985) we suggest that the underlying mechanism is the
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'Ca2+-induced release of Ca2+' (CIRC) with Ca2+ influx through Ca2+ channels as release trigger (see below). The Ca2+ release contributes a transient increase and decay of the [Ca2+]i change following the membrane depolarization which is called 'phasic' in the present work. It should be mentioned also, however, that Ca2+ influx generates a time-dependent [Ca2+]i change, as long as ICa activates and inactivates. Thus, the [Ca2+]i transients obtained with suppressed CIRC were not time independent but rose to a peak and slowly decayed (Figs 4, 5, 6 and 7). After several seconds, however, when ICa inactivation had become steady, Ca2+ influx occurred at a constant rate, and the sustained [Ca2+]j became time independent at a level where Ca2+ influx was balanced by Ca2+ extrusion. Although the results are clear in a qualitative way, they do not permit a quantitative dissection of the [Ca2+]i transient into a component due to Ca2+ release and a component due to Ca2+ influx. The difficulty arises because the [Ca2+]i transient also contains components of the redistribution of Ca2+; a simple subtraction of the [Ca2+]i transient recorded in the presence of the thapsigargin from the control recording would contain the contributions of suppressed release, suppressed reuptake plus those of stimulated Ca2+ extrusion. Since the components are not independent of each other a simple subtraction is not possible. For example, thapsigargin (as well as ryanodine) increased the 'resting' [Ca2+]i and due to Ca2+-induced inactivation of L-type Ca2+ channels the amplitude of ICa was diminished. In the case of caffeine, direct inhibition of the Ca2+ channels has been reported (Hughes, Hering & Bolton, 1990). Thus, when Ca2+ influx was reduced below control level by the above interventions, the contribution of control Ca2+ influx to the depolarization-induced [Ca2+]i transient is expected to be underestimated. The results of the present study may suggest that at 3-6 mm [Ca2+]0 and 36 °C with membrane potential steps to 0 mV approximately 70 % of the increment in [Ca2+]i is due to CIRC and only 30% due to Ca2+ influx. The relative importance of the two processes, however, depends on a variety of factors including the time after beginning of the depolarization. This value can increase to about 50% when ICa is increased by elevation in [Ca2+]o (Fig. 8) or by addition of a Ca2+ agonist (Figs 9 and 10). The relative importance of CIRC can decrease to values below 50% with submaximal activation, i.e. during graded responses, when the trigger of Ca2+ release is reduced. An example was given for depolarizing pulses to -30 mV, in the presence and absence of Bay K 8644 (Fig. 9B). The trigger for CIRC also diminishes when the duration of the depolarizing step was shortened to less than 300 ms (Ganitkevich & Isenberg, 1991). Thus, the question of the contribution of CIRC and Ca2+ influx in other experimental conditions, like during one or several action potentials, has to be evaluated in future. Compared to [Ca2+]i transients in cardiac preparations, where the role of CIRC is well established (Beuckelmann & Wier, 1988; Wendt-Galitelli & Isenberg, 1991), in smooth muscle cells under study the depolarization-induced [Ca2+]i transient peaked within approximately 300 ms (or even more) while in a cardiac specimen within about 20 ms. A recent study of reconstituted ryanodine receptors indicated that the release channel from vascular muscle has a similar [Ca2+]i sensitivity as the release channel from skeletal or cardiac SR (Herrmann-Frank, Darling & Meissner, 1991). If
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it is not the channel protein per se, then the ultrastructural organization of the SR could be responsible for the difference. For example, it is possible that some parts of SR in smooth muscle cells are located at different distances from the surface membrane than others. Activation of CIRC there is expected to occur with some diffusional delay; diffusion could be also hindered by the membranes of peripheral SR. This possibility is indirectly supported by the result that IK Ca' which is activated by Ca2+ ions close to the inner side of the surface membrane, peaked and fell faster than the global [Ca2+]i as it is monitored by the Indo- 1 (see Figs 1, 3A and 4A). The dye Indo-1 competes with native intracellular Ca2+ binding sites, e.g. of proteins. The concentration of the dye (100/tM) was low in comparison with the endogenous Ca2+ buffer, estimated to be > 1 mm (Becker, Walsh, Singer & Fay, 1988). Interventions which resulted in the increase of the resting [Ca2+]i are expected to saturate the endogenous Ca2+ buffer to a higher degree. Competition of these sites with the Indo-1 would be less, and the dye signal may rise at higher rate. Thus, the 'square-like' [Ca2]i transient that was recorded in the presence of ryanodine from a resting [Ca2+]i of 200 nm should not be extrapolated to the control. A similar argument holds true for the contribution of peak and sustained ICa to the [Ca2+]i change. At the beginning of the depolarization [Ca2+]i is low and a large amount of inflowing Ca2+ should be buffered. During the sustained level [Ca2+]i is elevated to 300-500 nm, part of the buffer should be saturated and the relative small variation Of ICa through non-inactivating L-type channels (about 20 pA, Imaizumi et al. 1989) can modulate [Ca2+]i more effectively. Thus, when the degree of Ca2+ binding is different, the time course of the [Ca2+]i should not be subtracted. We are grateful to Professor D. A. Eisner for his comments on the manuscript.
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