Inositol 1,4,5-trisphosphate releases intracellular Ca2+ in ...

Inositol 1,4,5=trisphosphate releases intracellular in permeabilized chick atria ANA-MARIA Department

VITES

AND

ACHILLES

Ca2+

PAPPANO

of Pharmacology, University of Connecticut Health Center, Farmington,

VITES, ANA-MARIA, AND ACHILLES PAPPANO. Inosit011,4,5trisphosphate releases intracellular Ca2+ in permeabilized chick atria. Am. J. Physiol. 258 (Heart Circ. Physiol. 27): Hl745Hl752, P990.-Inositol 1,4,5trisphosphate (IP3) and caffeine evoked transient, reversible, and concentration-dependent increases in tension in saponin-treated chick atria1 muscle. Contractures evoked by IP, and caffeine were detected in solutions with 70 PM EGTA at pCa 7.0. In the presence of 7 mM EGTA, neither IP, nor caffeine was able to evoke a contracture. Maximally effective concentrations of IP, (20 ,uM) and caffeine (20 mM) developed tensions to -44 and 83% of that elicited by pCa 5.0 (maximum tension = lOO%), respectively. The IPs- or caffeine-induced contractures were consistently reproduced when the sarcoplasmic reticulum (SR) had previously been loaded with calcium. Preexposure to caffeine suppressed the following IPx-induced response. When ryanodine (l-10 PM) was present throughout the SR-loading cycle, the responses to IP, and caffeine were prevented. However, when ryanodine was added after the SR was loaded with calcium, neither the response to IP, nor that to caffeine was affected. These results are consistent with the hypothesis that ryanodine inhibition requires prior activation of the SR calcium-release channel. It is concluded that both IP, and caffeine increased tension in the SR by releasing calcium from it. The effect of IP, is consistent with its messenger role as a calcium-mobilizing agent.

calcium release; cardiac sarcoplasmic phoinositides; ryanodine

reticulum;

caffeine; phos-

OF A PHOSPHOLIPASE C by a variety of chemical agents stimulates the hydrolysis of plasmalemma1 phosphatidylinositol4,5-bisphosphate into two messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 has been shown to serve as an intracellular messenger for calcium mobilization in various tissues, while DAG activates protein kinase C (12). Activation of the cardiac muscarinic and al-adrenergic receptors by an agonist induces a positive inotropic effect in the heart and stimulates the production of inositol phosphates, in particular IP3 (14, 20, 21, 23). In the case of muscarinic agonists the concentrations required are higher than those that inhibit adenosine 3’,5’-cyclic monophosphate (CAMP) formation (2, 27). Since it has been suggested that IP3 might serve as an intracellular messenger to mobilize calcium in heart, much of the work done with IP3 in cardiac tissue has focused on calcium mobilization. Sarcoplasmic reticulum (SR) vesicular preparations and permeabilized cells from cardiac tissue have been used to determine whether IP, can induce calcium release. Whether IP3 can release ACTIVATION

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calcium from cardiac SR is controversial because there is no agreement that IP, directly releases calcium (8, 13, 18, 19, 31). Therefore, we conducted experiments to ascertain whether IP3 could increase developed tension in cardiac tissue by releasing calcium from an intracellular pool. Since an elevation in cytoplasmic free calcium activates the myofilaments, causing an increase in developed tension, we used developed tension as a probe to detect the changes in cytoplasmic free calcium. Tension was recorded from very thin bundles of atria1 muscle permeabilized by previous treatment with saponin (19). We detected an IPS-sensitive calcium pool that could contribute to the generation of tension in cardiac tissue. Subsequently, we characterized the IP3-sensitive calcium pool by studying its interaction with ryanodine and caffeine, two agents that can diminish calcium stored in the SR (7, 10, 15). METHODS

Dissection and permeabilization of atria1 muscle fibers. Atria1 muscle fibers were dissected from the hearts of 2to &day-old chicks in Tyrode solution at room temperature (24°C). The Tyrode solution composition was (in mM) 137 NaCl, 12.0 NaHC03, 1.0 NaH2P04, 5.4 KCl, 1.0 MgC&, 1.8 CaC&, and 5.5 dextrose. The dissected fibers, 3-5 mm long and 120-220 pm in diameter, were placed in a tissue chamber (700 ~1). One end of the fiber bundle was pinned to the Sylgard-lined floor of the chamber, and the other end was attached to a force transducer via a glass capillary. The chamber was continuously perfused at 10 ml/min. Fibers were exposed to “relaxing” solution for -5 min before they were permeabilized by treatment with saponin (50 pg/ml for 40 min), which was added to the relaxing solution at 24°C. After saponin was washed out, the fiber was returned to relaxing solution, stretched, and allowed to equilibrate for at least 30 min. Saponin is a detergent that perforates preferentially the plasma membrane (30). The saponin treatment leaves functionally intact mitochondria and sarcoplasmic reticulum as described elsewhere (11, 28). The composition of the relaxing solution was (in mM) 3.17 MgCl,, 140.0 KCl, 25 imidazole, 4.22 K,ATP, 14 phosphocreatine, and 7 ethylene glycol-bis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), with 1.6 U/ ml creatine phosphokinase. The pH was adjusted to 7.2 with KOH, and the experiments were done at 24°C. pCa- tension curves. Permeabilized fibers were super-

0 1990 the American

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fused with test solutions containing various calcium concentrations. EGTA at 70 PM and 7 mM was used to buffer calcium in the test solutions that were prepared by adding the appropriate amount of CaClz. The free calcium concentrations are expressed as pCa (-loglo[ Ca”‘] ). The tension developed increased with increasing concentrations of calcium in the solution. Zero tension was taken as that obtained in the relaxing medium. Values of tension achieved at the various calcium concentrations were normalized relative to the maximum calcium-induced tension achieved by the fiber ( Tmax). Test solution composition was (in mM) 3.17 MgC12, 140 KCl, 25 imidazole, 4.22 KBATP, and 14.0 phosphocreatine, with 1.6 U/ml creatine phosphokinase. The appropriate amount of CaC12 was added to attain a desired free calcium concentration according to the association constants given by Fabiato and Fabiato (9). The amount of EGTA was either 7 mM or 70 PM. Solutions were buffered to pH 7.2 with KOH or HCl. SR “loading. fl To make the intracellular calcium stores as homogeneous as possible before each trial, we used a procedure based on that given by Nosek et al. (19) with some modifications. The permeabilized atria1 fibers were bathed in the following sequence of solutions: solution 1, place in relaxing solution for 5 min; solution 2, add 25 mM caffeine to solution 1 for 2 min to unload calcium from the SR; solution 3, return to solution 1 for 5 min to wash out caffeine; solution 4, load the SR with calcium by an 8 min exposure to pCa 6.5, 7 mM EGTA test solution; solution 5, place fibers in 70 ,uM EGTA test solution at pCa 7.0; this was the solution in which responsesto IP3 and caffeine were tested. In this medium, increments in cytoplasmic calcium cause an increase in developed tension. Solution 6, induced Tmaxby exposing the fibers to 70 PM test solution at pCa 5.0. A new cycle was then started by exposing the fiber to solution 1. The tension was readjusted (if necessary) to match the initial tension imposed on the permeabilized atria1 fiber in solution 1. In four preliminary experiments, we observed that the IP,- or caffeine-induced contractures progressively decreased when the muscle was not subjected to calcium unload-load cycles between exposures. Quantification of the drug-induced contractures. The IP3- and caffeine-induced developed tensions were compared and normalized relative to Tmax(solution 6). Drugs were tested in solution 5 at pCa 7.0 and [EGTA] 70 PM. Provided that the IP3- and caffeine-induced responses were normalized to Tmaxwithin each cycle, the transient rise in tension evoked by these agents was reproducible and consistent from four to seven cycles in a single fiber. In six fibers (n > 9 trials), we evaluated the contractures induced by 10 mM caffeine when the buffer (pH 7.2) was either 30 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) or 25 mM imidazole. For these experiments, the response (mean t SD) to caffeine averaged 54 t 6.6 and 62 t 4.9% Tmaxin TES and imidazole, respectively. Therefore, imidazole was an appropriate buffer under our experimental conditions. Sources of chemicals. IP3, caffeine, saponin, KCl, EGTA, imidazole, K2ATP, phosphocreatine, creatine phosphokinase, and TES were purchased from Sigma,

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St. Louis, MO. IP3 from Amersham, Arlington Heights, IL, was used in a few experiments; we did not detect any differences between sources. Most of the work described here was done with IP, purchased from Sigma; it contains two isomers, 80-90% l,4,5 isomer and the remaining primarily 2,4,5 isomer. Ryanodine was obtained from S. P. Penick, West Lynhurst, NJ. All the salts with the exception of KC1 were purchased from J. T. Baker, Phillipsburg, NJ. RESULTS

Sensitivity of permeabilized chick atria1 muscle to calcium. To compare our system with others, we determined the myofilament sensitivity to calcium in our preparation using 7 mM EGTA-buffered solutions. The developed tension was recorded at increasing concentrations of calcium, and its value was normalized relative to the maximal tension achievable by the fiber (see METHODS). An increase in developed tension occurred at pCa 7.0, reached half-maximum at -pCa 6.0, and maximum tension at -pCa 5.0 (data not shown). Since most of the experiments to detect developed tension upon drug exposure were performed in a lowEGTA buffering system (70 ,uM), we also determined the pCa-tension curve under these conditions. The pCatension curve in Fig. 1 shows that pCa 5.0 induces the maximum and pCa 6.2 the half-maximum tension in these fibers. The steepest slope of the pCa-tension relation (Fig. 1) is between pCa 7.0 and 5.5. Therefore a small change in cytoplasmic calcium concentration in this region of the curve can induce a large change in tension. This is the rationale for the use of pCa 7.0 and 70 PM EGTA in the experiments with IP3 and caffeine. Caffeine-induced tension transients. Caffeine induced a transient increase in tension in permeabilized atria1 muscle fibers bathed in 70 PM EGTA test solutions (Fig. 2 0 zw

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FIG. 1. pCa-tension curve in 70 PM EGTA-buffered test media. Permeabilized atria1 muscle fibers were exposed to increasing concentrations of free calcium in 70 ,uM EGTA-buffered media. Submaximal developed tensions are expressed as a % calcium-induced tension (T,,,) achieved in the 70 ,uM EGTA media in each run. Note that although the absolute value of Tmax decreased from one run to the next (lst, 339 mg; 3rd, 310 mg; 5th, 260 mg), sensitivity to calcium of myofilaments did not change with time. Latter is shown in this figure, where shape of curve is not modified after absolute values of tension were normalized relative to Tmax in each individual run. T,,, was usually achieved at pCa 5.0 (10m5 M) and was half-maximal at pCa = 6.2. Figure is average of 10 fibers (range 7-18 runs). Bars indicate SE.

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2, A and C). The presence of 7 mM EGTA prevented the caffeine-induced contracture, which indicates that an increase in cytoplasmic free calcium mediates the response (Fig. ZB). Caffeine-induced contractures were concentration dependent (Fig. 3). The maximum response was attained at 20 mM caffeine and was equivalent to 83 t 11% (SD) The lowest concentration at which caffeine inof Lw variably produced a detectable increase in tension was 1 mM. To determine whether a change in myofilament senT max

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sitivity to calcium could account for the responses observed, we compared the pCa-tension relationship in the presence and absence of caffeine (Fig. 4). Caffeine shifted the pCa-tension curve -0.3 units to the left, which indicates that a small portion of the response is due to an increase in myofilament sensitivity to calcium. IP3-induced tension transients. Like caffeine, IP3 induced a transient increase in tension when added in the presence of 70 FM EGTA at pCa 7.0 (Fig. 2, A and C). However, in the presence of 7 mM EGTA, IP, did not develop any tension, which indicates that IP3 evokes a transient rise in tension by increasing cytoplasmic free calcium (Fig. ZB). IP3-induced responses were concentration dependent, as shown in Fig. 5. A maximum response was observed at 20 PM IP3. Concentrations of IPB below 1 PM did not invariably produce a detectable response (Fig. 5). Note that the basal tension in 70 PM EGTA and pCa 7.0 averaged 8% of TmaX(Fig. 1). We also tested the effect of IP3 on the myofilament

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FIG. 2. EGTA (mM concn) completely abolishes IP3- and caffeineinduced contractures. Three consecutive runs of same fiber are shown where SR was loaded between runs. In each run, 10 PM IP, and 10 mM caffeine were applied. Vertical bar labeled T,,, indicates maximal tension induced by calcium in that run (pCa 5.0, 70 PM EGTA). A: drugs added in presence of 70 PM EGTA. B: drugs added in presence of 7 mM EGTA. EGTA (mM concn) buffered the calcium that mediates contractures. C: fibers are returned to 70 PM EGTA at pCa 7.0 as in A. Effect of high concentrations of EGTA is completely reversible.

PCs FIG. 4. Effects of caffeine on sensitivity of myofilaments to calcium. pCa-tension curves (as described in Fig. 1) were obtained in absence and presence of 10 mM caffeine. Relative and absolute developed tensions at a specific calcium concentration were examined. Caffeine (10 mM) had same effect in 4 different fibers (16 runs), a small depressive effect on absolute T,,,, reducing it to 86% of that in absence of caffeine. It also caused a slight increase in myofilament sensitivity to calcium by shifting pCaso 0.3 units higher.

f

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10 [CAFFEINE],

100 mM

3. Caffeine-induced transient increases in tension are concentration dependent. Various caffeine concentrations were added to permeabilized atria1 fibers in presence of 70 PM EGTA after SR was loaded. Responses obtained were similar to those shown in Fig. 2, A and C. Peak value of response was systematically normalized relative of caffeine that gave the maximum response to znax. Concentration (83% T,,,,) was -20 mM. Bars indicate SD. Results were obtained from 30 different fibers (range 4-25 runs). FIG.

VP31,PM 5. Concentration dependence of IPZ-induced tension transients. Various concentrations of IP3 were tested at pCa 7.0 and 70 FM EGTA. Responses similar to those shown in Fig. 2, A and C were obtained. Peak value of tension attained after IP3 addition was normalized relative to Tmax (pCa 5.0) of that individual run. Figure summarizes results obtained from 36 fibers (range 2-20 runs). Maximal response to IP3 (20 ,uM) reached a tension equivalent to 44% TmaX. Bars indicate SD. FIG.

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sensitivity to calcium. As shown in Fig. 6, IP3 (8 PM) did not affect the pCa-tension relationship. These results indicate that the responses to IP3 are due to an increase in cytoplasmic calcium rather than to a change in the sensitivity of the myofilaments to calcium. Interaction between IPs and caffeine calcium pools. In a series of 25 experiments, we tested the possibility of an interaction between IP3 and caffeine. For these experiments, the SR was not subjected to the loading procedure (see METHODS) between exposures to either agent. As shown in Fig. 7A, exposure of the fiber to 20 PM IP3 evoked a transient increase of developed tension (46% T,,,) that largely dissipated within 1 min. At 4-5 min after introduction of IP3, 20 mM caffeine elicited a tension transient that attained 83% Tmax. When the sequence was reversed, addition of 20 mM caffeine raised the fiber developed tension to 88% Tmax.After 4 min in the presence of caffeine, the contracture in response to 20 PM IP,? was suppressed (Fig. 7B). This indicates that caffeine depletes calcium from an IP,?-sensitive calcium pool, presumably the SR. Effects of ryanodine. Ryanodine is thought to deplete SR calcium by locking the SR calcium-release channel in a low-conductance open state (10, 24). Ryanodine (low5 M) did not affect the immediate caffeine- or IP3induced tension transients (see Figs. 8B and 9B). In similar experiments to those shown in Figs. 8B and 9B, the responses remained unaffected even when ryanodine was present for >30 min. However, when ryanodine was present throughout the SR unloading-loading procedure (Figs. 8C and 9C), the IP3- and caffeine-induced responseswere prevented. Spontaneous fluctuations in tension. Spontaneous fluctuations in tension were observed in 48 of 50 fibers at 70 PM EGTA and pCa between 7.0 and 5.5 and did not require the SR to be loaded with calcium (see METHODS). Their amplitude, shape, and frequency varied from muscle to muscle. An example is illustrated in Fig. 10, where the spontaneous fluctuations of two different fibers are shown. In all of the experiments with caffeine, the spontaneous fluctuations in tension were reversibly suppressed. We determined the concentration dependence for caffeine-induced suppression in four experiments; an @ + 8 pM IP3

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IP3 FIG. 7. IP:, and caffeine share same calcium pool. Interaction shown between IP, and caffeine responses in 2 subsequent runs in same fiber after SR was appropriately loaded between runs. A: fiber exposed to 20 ,uM IP, (maximal concentration; see Fig. 5) in pCa 7.0 and 70 PM EGTA. After IPs-induced response was over, 20 mM caffeine (maximal concentration; see Fig. 4) was added. B: 20 mM caffeine added, then 20 PM IP3. In this order caffeine almost abolished subsequent response maximum tension obtained by to 20 /JM IP:3. T,,,- labeled bar indicates exposing fiber to lo-” M calcium immediately after responses were recorded. A, IP3 and caffeine increased tension by 43 and 75% of T,,, (165 mg), respectively. On 2nd run (B) IP, increased tension by only 8% of T,,, by caffeine by 72% (133 mg).

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FIG. 6. IP:, does not affect pCa-tension relationship. pCa-tension curves were obtained in presence and absence of 8 PM IPS. IP3 affected neither relative nor absolute values of tension in fibers. Figure summarizes results from 2 fibers (4 runs in presence of IP3 and 6 runs in its absence).

FIG. 8. Effect of ryanodine on the caffeine-induced responses. Ryanodine must be present throughout one loading cycle to block response to caffeine. A: control response to 20 mM caffeine. B: ryanodine up to 10e5 M had no effect on immediate caffeine response when added to pCa 7.0 and 70 PM EGTA medium. In similar experiments in which ryanodine was exposed for >30 min, caffeine-induced responses remained unaffected. C: after ryanodine had been present throughout one cycle, caffeine-induced response was prevented. Tmax in A, B, and C was 136, 150, and 150 mg, respectively. Records are 3 consecutive runs in same fiber, and these results were reproduced in 6 other fibers.

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IP3 FIG. 9. Effect of ryanodine on IPs-induced responses. Records show activity-dependent effect of ryanodine on IPs-induced calcium-release mechanism(s). A: control response to 20 PM IP,. 23: low5 M ryanodine added to pCa 7.0 and 70 ,uM EGTA, 4 min before exposure to 20 PM IP, (bar). Ryanodine did not affect immediate IP3-induced response even when ryanodine was present for ~30 min before IPB exposure. However, when ryanodine was present throughout soZutions 1-5 (see METHODS), IPs-induced response was suppressed, as shown in C. Absolute values of T,,, for runs in A, B, and C are 264, 240, and 255 mg, respectively. Records show 3 consecutive runs of same fiber. Results were reproduced in 4 other fibers.

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FIG. 10. Effect of increasing concentrations of caffeine and ryanodine on spontaneous fluctuations in tension. Records show spontaneous fluctuations in tension in 2 different fibers. Vertical calibration bar is 25 mg; horizontal bar is 1 min. A: typical record of permeabilized fiber in medium buffered with 70 PM EGTA at pCa 7.0. Fiber was exposed to increasing concentrations of caffeine. Recordings show steady-state response to specific caffeine concentration. lo-” M caffeine completely abolished spontaneous fluctuations in tension. Effect is reversible after caffeine is washed out (WO, bottom trace). B: record from different fiber under same conditions as A, but fiber was exposed to increasing concentrations of ryanodine. Records shown are taken after steadystate response to given concentration of ryanodine was achieved. Ryanodine concentrations of lo-” M prevented spontaneous fluctuations in tension after 5 min exposure, whereas lo-” M ryanodine had same effect in 1 min. Effect of ryanodine was irreversible after washout.

at pCa 5.8 and 5.3, respectively (7, 17). Nosek et al. (19), who used 7 mM EGTA test solutions as we did, reported 50% of Tmaxat pCa 5.8, whereas we found it to be pCa example is shown in Fig. 1OA. The spontaneous fluctua6.0. In addition to the high EGTA-buffered solutions (7 tions were gradually suppressed by increasing concentrasolutions tions of caffeine; complete suppression occurred in 10 mM), we also used very low EGTA-buffered (70 PM) to test the actions of IP, and caffeine. The pCamM caffeine. The effect was reversed after a few minutes tension relationship in the 70 PM EGTA test solutions of caffeine washout (Fig. lOA). As shown in Fig. IOB, exposing the tissue to increasing concentrations of ry- yielded 50% of Tmaxat pCa 6.2. Nosek et al. (19) found that IPS enhanced the caffeineanodine also reduced the spontaneous fluctuations in tension. This effect was not reversed after 60 min wash- induced response and the spontaneous fluctuations in out of ryanodine (8 of 8 fibers). Exposure to 10s6 M saponin-treated guinea pig papillary muscle. By contrast, we observed a concentration-dependent increase in tenryanodine completely abolished the spontaneous activity sion in response to IPS, but we did not detect any effect in 5 min whereas 10B5 M ryanodine had the same effect of IP3 on the spontaneous fluctuations in tension. Therein 4 min (not shown). fore, some of our results extend and others differ from those described by others (19). Although we do not have DISCUSSION an explanation for these differences, it is noteworthy The principal experimental finding is that IP3 induced that we found that IP3 by itself increased developed transient increases of tension in hyperpermeable atria1 tension in saponin-treated atria1 muscle fibers. Although muscle. Our evidence indicates that IP3 increases devel- we used a protocol for calcium loading similar to that described by Nosek et al. (19), the loading time was 8 oped tension by releasing calcium from the SR. This preparation is a sensitive and reliable system not only min in our experiments compared with 3 min in theirs. for detecting and quantifying IPs-induced responses but Fabiato (8) observed IP3-induced calcium release and an also for elucidating a model for the IP3-induced calciumincrease in developed tension in mechanically skinned release mechanism. Before a discussion of these findings, rat ventricular cells. Our results agree with his findings. it is helpful to compare the properties of our preparation Hirata et al. (13) found that IP3 (5 PM) released calcium with those of others. The myofilament sensitivity to from canine ventricular SR vesicles, whereas Movesesian calcium of our preparation in 7 mM EGTA is within the et al. (18) reported that IP3 (up to 50 PM) was not an ranges described by others. Fabiato (7) and Miller and effective calcium-release agent either in permeabilized Smith (17) used 10 mM EGTA and obtained 50% of Tmax rat cardiac myocytes or in isolated SR. Fabiato (8) ob-

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served that the tension increased from 3 to 10-B% of Tmax when IPs was increased from 2 to 30 PM in single skinned rat ventricular cells. In our preparation the tension increased from 19 to 44% of Tmax when IP, was increased from 1 to 20 PM. We used saponin-treated multicellular preparations, in which the slow diffusion of the stimulating agent into the fiber makes the individual cells in the bundle respond in a temporarily dispersed manner. This dispersed response induces a smaller, slower increase in tension that can consequently result in an overestimation of the concentration at which maximum response is attained in our preparation. Considering the limitations existing in a multicellular preparation, the concentrations of IP3 we used are in very close agreement with those used by Fabiato (8) in single ventricular cells, where these problems are greatly reduced. Our preparation is sensitive to IP3 in a concentration range similar to those used to induce physiological responses in muscle (29) and nonmuscle cells (1). The levels of free calcium reached at maximal concentrations of IP3 were high enough to induce 44% T,,,. From Fig. 1, 44% Tmax was achieved at free calcium concentrations of 5 X 10v7 M or pCa 6.3. We did not detect any change in the sensitivity of the myofilaments to calcium in these fibers in the presence of IPs (Fig. 6). This result is in agreement with Fabiato (8), who used up to 200 PM IP3, and Nosek et al. (19), who used 30 PM IP3. Therefore, the IP3 response is not due to a change in sensitivity of the myofilaments to calcium but to the contracture of the myofilaments in response to the increase in free calcium. To obtain consistent responses, we loaded a calcium store within the muscle, which is presumably the SR. This, in addition to the fact that the response was absent in media with large calcium buffering capacity (7 mM EGTA), suggests that calcium released from a cytoplasmic store mediates the IPS-induced increase in tension. The experiments shown in Fig. 2 indicate that caffeine, like IP3, induces an increase in tension through a cascade pathway that requires the elevation of cytoplasmic calcium levels. This cascade is occluded by the presence of 7 mM EGTA (Fig. ZB). Like IP3, caffeine induced uniform responses after the SR was loaded with calcium (see METHODS). These observations indicate that caffeine and IP3 act by releasing calcium and increasing cytoplasmic free calcium. The caffeine-induced responses shown in Fig. 2 are concentration dependent (Fig. 3). Maximal concentrations of caffeine (20 mM) generate an average peak response of 83% Tmax. The concentrations of caffeine we used are within the range used in other muscle tissues (5, 15, 19, 25, 26). In addition, caffeine had a small effect on the myofilaments’ sensitivity to calcium, increasing the sensitivity by 0.3 pCa units (Fig. 4). An effect of caffeine on the myofilaments has been previously reported (4, 6). Therefore, the increase in tension induced by caffeine is mostly due to an increase in cytoplasmic calcium. At maximally effective concentrations, the caffeine-induced contracture was longer than that induced by IP,?. The different durations of such contractures may be due to differences in the amount of calcium released

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by IP3 and caffeine. However, this interpretation is constrained by several limitations, including diffusion in a multicellular preparation, increased myofilament sensitivity to calcium with caffeine but not IP,?, and enzymatic degradation of IP3 in the muscle. We asked whether the store(s) from which caffeine releases calcium is the same as that acted upon by IPZ. Caffeine-induced contractures were greatly diminished by previous exposure to caffeine itself. This effect was also true for IP3-induced contractures. This effect had been previously demonstrated for caffeine in skeletal muscle, where successive exposures to the same concentration of caffeine without loading the SR between trials resulted in the reduction of the second induced contracture (5). We observed that preexposure to maximum concentrations of caffeine greatly diminished the subsequent response to maximum concentrations of IP,. This suggests that the IPy-sensitive calcium pool becomes unavailable after prolonged exposure to high concentrations of caffeine (Fig. 7). These experiments indicate that IP3 acts on a caffeine-sensitive calcium pool, which is presumably the cardiac SR. These results show that IP3 plays a similar role in cardiac muscle as it does in other muscles and nonmuscle cells, where it may act as a messenger to mobilize calcium from the SR and endoplasmic reticulum, respectively (1, 29). In addition to the IP3- and caffeine-induced contractures, we observed small spontaneous fluctuations in tension. They have been associated with spontaneous bursts of calcium released from the SR (3, 7, 16, 19). We observed them even in the absence of SR calcium loading, as did Fabiato (7). They were present between pCa 7.0 and 5.5, in the low EGTA-buffered solutions. The spontaneous fluctuations in our experiments were irreversibly prevented by ryanodine and reversibly by caffeine. Both agents acted in a concentration-dependent fashion (Fig. 10, A and B). Threshold concentration for caffeine (10m7 M) slightly diminished the amplitude of the fluctuations, whereas 10m2 M caffeine completely prevented them. Ryanodine concentrations of 10m7 M reduced the frequency and amplitude, and 10m6 M ryanodine completely abolished them. These observations are in close agreement with those described by others (16). In intact rat ventricular cells, caffeine concentrations between 10s4 and 10v2 M increased the frequency and reduced the amplitude of the oscillations, whereas ryanodine (lo-’ to 10B7 M) decreased both the frequency and amplitude of the spontaneous fluctuations in tension (3). We observed similar effects for caffeine and ryanodine. The concentrations of ryanodine were one order of magnitude higher in our preparations. The difference may depend on the fact that we used permeabilized muscle and they used intact cells. We did not detect an effect of IP3 on the spontaneous fluctuations in tension as described by others (8, 19). The different results may be related to the different cardiac tissues used in their experiments (guinea pig and rat ventricular muscle) and in ours (chick atria1 muscle). The frequency of the spontaneous contractures (1-4/min) we observed is lower than that reported by other investigators (8, 19). We may have missed low-amplitude, high-frequency contractures be-

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cause of the noise inherent in the force transducer. Provided that the effect of IP, on the spontaneous contractures is mediated by calcium, the increase in calcium evoked by IP, is quite brief relative to the frequency of the spontaneous contractures. This may account for our not observing a calcium-mediated action of IP3 on the spontaneous contractures. We found a remarkable difference between the actions of ryanodine on the spontaneous fluctuations in tension compared with the IP3- and caffeine-induced responses. When ryanodine (l-10 PM) was added to the test media (pCa 7.0 and 70 ,uM EGTA), it suppressed the spontaneous oscillations in tension within 5 min (see Fig. 8B), whereas the immediate IP3- and caffeine-induced contractures remained unaffected (Figs. 8B and 9B). However, when ryanodine was present during the calcium unloading-l .oading cycle (solutions l-5), the responses to caffeine and IP3 were largely suppressed (Figs. 8C and 9C). This phenomenon has also been reported for caffeine in rabbit papillary and skeletal muscle (25, 26) and in rabbit smooth muscle (15). Fabiato (7) also found that ryanodine had no immediate effect on the calcium-induced calcium release tension transient but that it decreased the subsequent responses in a progressive manner in rat ventricular cells. Altogether, these findings are consistent with the hypothesis that ryanodine binds to the SR calcium-release channel in its “open” or “active” state (10, 25, 26). This view does not exclude the possibility that the binding of ryanodine to its receptor requires calcium (15). An interpretation for the unequal action of ryanodine on the spontaneous fluctuations in tension and the druginduced responses may be the existence of a distinct calcium pool for the spontaneous fluctuations in tension. This interpretation is supported by the fact that even when ryanodine was present in the test solution for ~30 min, the IP3 and caffeine responses remained unaffected (not shown). Another possible interpretation is the existence of two distinct calcium-release channels in the SR dur results indicate that 1) caffeine reduces the response to subsequent exposure to IP3 (Fig. 7), 2) reproducible responses to a specific concentration of either IP3 or caffeine were obtained only after the SR was loaded with calcium, 3) high concentrations of EGTA prevent both IPS- and caffeine-induced contrac tures (Fig. 2), and 4) ryanodine abolished the responses generated by caffeine or IP3 in an activity-dependent fashion (Figs. 8 and 9). The similarity of caffeine and IP3 as calciummobilizing agents and their interaction indicate that IP3 acts on a caffeine-sensitive calcium pool, the SR. Activation of al-adrenoceptor and muscarinic agonist receptors increase the IP3 levels and induce a positive inotropic effect in the heart (2, 14, 21, 23, 27, 29). Our results show that IP3 by itself acts as a calcium-mobilizing agent that increases tension in cardiac muscle at concentrations similar to those reported in other muscle tissues (29). These increases in tension are not a result of an effect of IP, on the sensitivity of the myofilaments to calcium. The pool of calcium that mediates the IP3 response is shared bv caffeine and is presumablv the SR.

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IN

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Our results are consistent with the hypothesis that mobilization of intracellular free calcium by IP, is involved in inotropic action of muscarinic and cul-adrenoceptor agonists in the heart. This work was supported by National Heart, Lung, and Blood Institute Grant HL-13339. This work has been submitted by A. M. Vites to the Graduate School of Arts and Sciences, University of Connecticut, in partial fulfillment of the requirements for the doctoral degree. Address reprint requests to A. J. Pappano. Received

30 May

1989; accepted

in final

form

18 January

1990.

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