Intraluminal Ca2+ Dependence of Ca2+ and Ryanodine-mediated ...

THEJOURNALOF BIOLOGICAL CHEMISTRY

Vol. 267, No. 29, Isaue of October 15, pp. 20850-20656,1992

0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Printed in U.S.A.

Intraluminal Ca2+ Dependence of Ca2+and Ryanodine-mediated Regulation of Skeletal Muscle Sarcoplasmic ReticulumCa2+Release* (Received for publication, March 5, 1992)

James S. C. GilchristSO,Angelo N. Belcastroll, and Sidney Kat211 From the $Faculty of Graduate Studies, TDivision of Kinesiology, Faculty of Education, and 11 Faculty of Pharmaceutical Sciences, university of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dualwavelength spectroscopy of Ca’+:antipyrylazo I11 difference absorbance, the intraluminal Ca2+dependence of ryanodine and Ca2+-inducedCa2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+uptake (a)increased the intraluminal Ca2+sensitivity of CICR and ( b )stimulated spontaneous Ca2+release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+load. Ryanodine also inhibited subsequentCICR after reaccumulationof Ca2+released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+binding states of an intraluminal Ca2+compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+release channel.

by ruthenium red and millimolar Mg2+ (Kim et al., 1983; Ikemoto et al., 1984; Meissner, 1984; Meissner et al., 1986). These effectors interact directly with the cytosolic region of the release channel (Liu et al., 1989). Regenerative type Ca2+ release mechanisms (both Ca2+- and caffeine-stimulated) have also been shown to exhibit dependence upon threshold filling of an intraluminal releasable Ca2+ compartment. This was demonstrated in earlier studies with skeletal skinned fibers (for review see Endo (1977)) and more recently with “heavy” SR vesicles (HSR) referable to the SR cisternae (Ohnishi, 1979, Nelson and Nelson, 1990). Ikemoto and co-workers (Ikemoto et al., 1989b, 1991) have shown that the rate and extent of caffeine-induced Ca2+ release maybe regulated through an intimate Ca2+-mediated structural andfunctional association of calsequestrin with the ryanodine receptor (see Collins et al. (1990)). In native vesicles, structural states of the release channeland Ca2+ release rateconstants were dependent upon the occupation of calsequestrin by intraluminal Ca2+ (Ikemoto et al., 1989b). Calsequestrin removal reversibly abolished the intraluminal Ca2+ transient and Ca2+ release (Ikemoto et al., 1989b, 1991). Indirect evidence suggests that the Ca2+ release channel states may, conversely, regulate the association of Ca2+with the intraluminal releasable Ca2+ compartment. Theplant alkaloid ryanodine, which binds specifically to the release The triggering of skeletal muscle contraction results from channel (Pessah et al., 1985) with both high and low affinity the rapid release of Ca2+ to the cytosol from the cisternal (Lai et al., 1989), was shown to deplete SR Ca2+in smooth lumen of sarcoplasmic reticulum (SR)’ membranes (see Ike(Ashida et al., 1988; Ito et al., 1986; Iino et al., 1988) and mot0 et al. (1989a) and Lai and Meissner (1989)). The precise cardiac muscle (Hansford and Lakatta, 1987; Hunter et al., trigger mechanism for SR Ca2+release, in response to t-tubule 1983). Whether this effect of ryanodine in native SR memdepolarization, is unknown. Several hypotheses have proposed branes is due to formation of a long-lasting subconductance that Ca2+release is stimulated through electrogenic (Schneistate of the Ca2+channel (Rousseau et al., 1987) and/or derand Chandler, 1973) and/or regenerative ( i e . Ca2+-ininhibition of intraluminal Ca2+binding is unclear. However, duced Ca’+ release or CICR) transduction processes (Ford we observed in HSR vesicles that elevated M e , in addition and Podolsky, 1970; Fabiato, 1989; Nesterov, 1988). to reducing the extraluminal Ca2+ sensitivity of CICR, inCa2+release is activated by millimolar adenine nucleotides for Ca2+ release (ATP, AMP-PCP, AMP) and micromolar Ca2+and inhibited creased the intraluminalCa2+threshold (Gilchrist et al., 1990). These preliminary observations sug* This work was supported by grants from the British Columbia gested that extraluminal effector modification of Ca2+channel and Yukon Heart and Stroke Foundation (to S. K ) and the Natural states may regulate the association/ dissociation of Ca2+ Science and Engineering Research Council of Canada (to A. N. B). within the intraluminal Ca2+compartment. In this study, we The costs of publication of this article were defrayed in part by the demonstrate that the action of ryanodine upon HSR Ca2+ payment of page charges. This article must therefore be hereby release is highly sensitive to the filling of an intraluminal marked “advertisement” in accordance with 18 U.S.C. Section 1734 Ca2+compartment, presumably calsequestrin. Depending solely to indicate this fact. 5 Supported by a British Columbia and Yukon Heart and Stroke upon the Ca2+-occupiedstate of the intraluminal compartFoundation Research Traineeship. Current address: Div. of Cardio- ment, bothactivatory and inhibitory effects of ryanodine upon vascular Sciences, St. Boniface General Hospital Research Centre, HSR Ca2+release can be demonstrated. These resultsprovide Faculty of Medicine, University of Manitoba, 351 Tache Ave., Win- evidence that state transitions of the Ca2+release channel nipeg, Manitoba, Canada R2H 2A6. ’ The abbreviations used are: SR, sarcoplasmic reticulum; AMP- and calsequestrin are interdependentlycoupled. AP 111, antipyrylazo 111; PCP, adenosine 5’-(/3,y-imino)triphosphate; CICR, calcium-induced calcium release; CP, creatine phosphate; DTT, dithiothreitol;HSR, heavy sarcoplasmic reticulum; PIPES, 1,4,-piperazinediethanesulfonicacid.

MATERIALSANDMETHODS

Isolation of HSR Membranes-HSR membranes were prepared from fast twitch rabbit skeletal muscle using the buffer systems

20850

Intraluminal Calcium and described by Chu et al. (1986) with several modifications. Back and hindlimb musclewas rapidly excised, trimmed of excess fat and connective tissue, and placed in 0.9% saline on ice within 10 min. Muscle was cut and weighed into 20-g portions and was immediately freeze-clamped in liquid Nz between aluminium tongs into 20-g discs, wrapped in aluminium foil, and stored at -70 "C. Frozen muscle was ground to a fine powder under liquid nitrogen using a porcelain pestle and mortar packed in ice. The powdered material was added to five volumes (w/v) of homogenization buffer containing 300 mM sucrose, 20 mM imidazole, 0.5 mM EGTA, 1 mM DTT, 2 mM phenylmethylsulfonyl fluoride, 10 pM leupeptin (pH 7.4) and was thawed (0-5 "C) with constant stirring. The protein slurry was homogenized in a 125ml Waring Blendor bottle a t slow speed (15,000 rpm) for two 40-s bursts with a 40-s interval. The homogenates were combined and were centrifuged for 10 min in a Beckman JA-14 rotor (Beckman Instruments, Palo Alto,CA) at 11,500 rpm. The supernatant was decanted and filtered through 4-ply cheesecloth and centrifuged at 45,000 rpm in a Beckman 50.2 rotor for 90 min. Pelleted material was suspended in homogenization buffer to 40 mg.ml" and centrifuged at 27,000 rpm (Beckman SW 28 rotor) for 16 h through a linear 25-45% (w/w) sucrose gradient containing 5 mM imidazole, 1 mM DTT, 2 mM phenylmethylsulfonyl fluoride, 10 g~ leupeptin (pH 7.4). HSR membranes enriched in ryanodine receptor protein were collected from the 38-40% region, slowly diluted (1:4) to 10% sucrose with buffer containing 150 mM KC1 and 20 mM imidazole, pelleted as above, and resuspended in homogenization buffer containing 10 p M leupeptin and 1 mM DTT toa final concentration of 40-50 mg.ml". In some preparations, 150 mM KC1was omitted from the dilution buffer. All procedures were conducted in the cold room and, typically, 200 g (10discs) of muscle were used per isolation. Assay of HSR Calcium Transport-Spectroscopic determinations of HSR Caz+ uptake and Caz+release were monitored in the dualwavelength mode of the SLMAminco DW2Cspectrophotometer with a recording wavelength pair of 675-790 nm as described (Gilchrist et al., 1990). HSR vesicles from freshly thawed stock (40-50 mg.ml") were preincubated to a final concentration of 1 mg. ml" for 3 min in 2.75 ml of transport media containing 300 mM sucrose, 100 mM KCI, 20 mM PIPES (pH7.0), 5 mM MgCI,, 5-20 mM phosphocreatine, 12.5 units.ml" creatine phosphokinase (EC 2.7.3.2; from rabbit skeletal muscle), 50 p~ AP 111 (25 "C) and,various concentrations of Ca2+as indicated in the figure legends. Ca2+uptake was initiated by addition of stock 50 mM Mg.ATP toa final concentration of 1 mM. Mg.ATP and other reagents were introduced to thesample compartment with a Unimetrics microliter syringe through a customized polystyrene diaphragm, which completely excluded the entry of external light upon initiation of and during Ca2+transport. Radiometric assay of Ca2+transport was performed using an identical buffer system to that described above except for the omission of AP 111 and use of '%az+ (50,000 dpm.nmo1-'). Ca2+ uptakeby HSR vesicles (1 mg.ml") was initiated by addition of Mg. ATP to a final concentration of 1 mM. At various time intervals, aliquots were filtered across 0.45-pm HAWP-type filters (see figure legends). Filters were then rinsed with 10 volumes of ice-cold Ca2+release-inhibition buffer containing 300 mM sucrose, 100 mM KCI, 20 mM PIPES (pH 7.0), 100 g~ EGTA, 5 mM MgC12, 50 p~ ruthenium red. Filters were air-dried and radioactivity on the filters determined by liquid scintillation using a Packard Tricarb 4530 liquid scintillation counter. Determination of Inorganic Phosphate-Inorganic phosphate accumulated by HSR vesicles was assayed spectroscopically by a modification of the procedure described by Raess and Vincenzi (1980). Nitrocellulose filters (0.45 gm HAWP-type, 13-mm diameter) containing trapped HSR vesicles were incubated overnight in 600 pl of 6.7% SDS contained within Eppendorf microcentrifuge tubes (see figure legends). Filters were pelleted at 14,000 X g for 30 min, and the supernatantwas aspirated into separate tubes. Color development was initiated by the sequential addition of 200 pl of 9% (w/v) ascorbic acid and 200 p1 of 1.25% (w/v) ammonium molybdate in 6.5% H,S04 (v/v). Absorbance at 660 nmwas recorded after 30 min of color development with KzHP04employed as a standard. Ryanodine Purification-Commercially available ryanodine was purchased from Agri-Systems International as a tan-colored powder (lot 8E09). This batch, which was poorly soluble above 5 mM in 10% methanol, was further purified by flash chromatography (see Ruest et al. (1985)). Ryanodine (50 mg) was dissolved in 3 ml of chloroform and was applied to a glass column (12 X 150 mm) packed with Silica Gel 60 (230-400 mesh). Ryanodine was eluted with 40 ml of CHCI,, MeOH, 40% aqueous CH3NH2(90:101.5) and identified with formation of a brown chromophore on heated HaS04-sprayed TLCplates

S R Calcium Release

2085 1

(Whatman 250 p M layer 4420 222). Fractions were pooledand vacuum dried with formation of a colorless translucent ryanodine residue, which was suspended in distilled, deionized H 2 0 to a final concentration of 20 mM.This procedure resulted in 50% recovery of the starting material weight. PHlRyanodim Bir~ding-[~H]Ryanodine (60 Ci/mmol) was purchased from Du Pont and used without further purification. HSR vesicles (400 pg. ml" final concentration) were incubated at 22 'C for 24 h in media containing 300 mM sucrose, 150 mM KCI, 5 mM AMP, 100 p M ca2+,100 p M leupeptin, 8 nM to 1 mM [3H]ryanodine,and 50 mM HEPES (pH 7.4). Stock [3H]ryanodine was added to a final concentration of 8 nM with increasing concentrations of total ryanodine effected by the addition of unlabeled ryanodine. 100-pl aliquots were vacuum-filtered across 0.45-pm (13-mm diameter) nitrocellulose filters, which were rinsed with 10 volumes of ice-cold buffer containing 300 mM sucrose, 150 mM KCl, and 50 mM HEPES (pH 7.4). Radioactivity remaining on air-dried filters was determined by counting in a Packhard Tricarb 4530 liquid scintillation counter. Specific binding was obtained by assuming nonspecific [3H]ryanodinebinding to be a constant proportion of total binding relative to [3H]ryanodine binding observed at 1 mM ryanodine. RESULTS

In agreement with earlier observations (Campbell et al., 1980), Fig. 1 (trace A ) shows that HSR vesicles, isolated in

the absence of KC1 containing buffers, retain a releasable ImM MgAW

FIG. 1. Spectroscopic determination of the release and reuptake of endogenous calcium by HSR vesicles. Ca2+uptake was monitored by dual-wavelength spectroscopy of Ca2+:AP111 difference absorbance at a 675 nm sample wavelength and a 790nm reference wavelength. HSR vesicles (1 mg.ml" final concentration) prepared in the absence of 150 mM KC1 washing were added (thin arrow) to the measuring cuvette directly into transport buffer containing 300 mM sucrose, 100 mM KCl, 20 mM PIPES (pH 7.0), 1 mM MgCI,, 5 mM CP, 12.5 units.ml" creatine phosphokinase, and 50 p~ AP 111. Vesicles were preincubated a t 25 'C until difference absorbance traces reached a plateau. Ca2+uptake was initiated by addition of 1 mM Mg.ATP to the cuvette (intermediate arrow). In A , accumulated Ca2+was released by addition of 10 p M A23187 to thecuvette (arrowhead). In traces B and C, 50 p~ ruthenium red was added to transport buffer before and after, respectively, the addition of vesicles. In D,vesicles were prewashed with 150 mM KCI. Downward excursions of the Ca2+:API11 difference absorbance trace indicate Ca2+ uptake, whereas Ca2+release is represented by upward excursions of the trace. The scales for time and Ca2+are as indicated. This experiment wasemployed as a test for each HSR preparation and is representative of in excess of 30 HSR membrane isolations prepared with and without KC1 washing.

20852

Intraluminal Calcium and SR Calcium Release

endogenous intraluminal Ca2+pool (80-90 nmol of Ca2+.mg" HSR). The released Ca2+ was reaccumulated triphasically after addition of 1 mM Mg-ATP to the cuvette. Subsequent collapse of the outward Ca2+gradient was observed after 10 PM A23187 addition. The presence of 50 p~ ruthenium red in the cuvette, prior to HSR addition, partially blocked the efflux of endogenous Ca2+(trace B ) . The Ca2+available for reaccumulation was consequently reduced. Addition of 50 p~ ruthenium red after passive Ca2+efflux led to the appearance of a single fast phase of ATP-dependent Ca2+uptake (trace C). Diminished slow phase Ca2+uptake is probably due to inhibition of Ca2+efflux through the Ca2+release channel. Vesicles prewashed with 150 mM KC1 did not, however, release endogenous Ca2+, eitherduring passive incubation or after A23187 addition (trace D ) . Thus KC1 washing of vesicles effectively displaced endogenous Ca2+. The HSR intraluminal Ca2+ dependence of Ca2+-induced Ca2+release (CICR), from vesicles prewashed with 150 mM KC1, isshown in Fig. 2. Vesicleswere preincubated with varying concentrations of exogenous Ca2+.After initial Ca2+ uptake with 1 mM Mg.ATP addition to the cuvette, vesicles were further loaded with 10 p M Ca2+pulses. Trace A shows that 10 p~ pulses of Ca2+(-5 p~ free), added subsequent to the initial uptake, were rapidly accumulated ( 2 0 0 nmol .mg HSR" min") up to an intraluminal Ca2+load of 80 nmol. mg". With another pulse addition of 10 p~ Ca", sub-maximal CICR wasobserved. This wasfollowedby maximal CICR after accumulation of 100 nmol of Ca2+.mg-l HSR protein. Observation of submaximal CICRwas routinely observed within a range of trigger Ca2+between 7.5 and 40 p~ Ca2+(320 p M free Ca2+)at intraluminal ca2+loads above 60 nmol. mg". With 5 p~ total trigger Ca2+ andbelow, partial releases ImM Mg.ATP

t

t

were abolished with observation solely of maximal CICR (see Fig. 4, trace D ) . As initial Ca2+was incremented by 10 pM (Fig. 2, truces BD ) , the amount of pulse Ca2+ required to elicit CICR was reduced by a corresponding amount. In all cases, maximal CICR was followed bya biphasic reaccumulation of virtually all extravesicular Ca2+. An initial slow phase (5-10 nmol. mg" .min") preceded a secondary intermediate phase (30-40 nmol. mg" .min-l), attributed to sustained release channel opening and spontaneous closing, respectively (Morii et ul., 1985).At elevated initial Ca2+loads (traces F and G),a similar biphasic uptake of Ca2+was observed subsequent to therapid accumulation of an initial 55-65 p M Ca2+.At higher initial extravesicular Ca2+ loads, the rate of secondary slow phase Ca2+ uptakeprogressively decreased (not shown). These data indicate that filling of an intraluminal Ca2+pool (>60 nmol. mg"), during ATP-dependent Ca2+ accumulation, is necessary for extravesicular Ca2+-stimulated Ca2+ channel opening. Depending upon the experimental manipulation of the relative distribution of extra- and intravesicular Ca2+, channel opening can be manifest as either( a )triggered release of Ca2+ down a concentrationgradient or ( b ) appearance of slow phase Ca2+accumulation. In Fig. 3 the effect of vesicle preincubation with ryanodine prior to addition of Mg. ATP was examined. Although a standard 5-min preincubation with ryanodine was employed, these same effects of ryanodine could also be observed almost immediately after addition of the drug. In the presence of 110 pM ryanodine, rapid Ca2+ uptakewas observed with slight decreases in the intraluminalCa2+ requirement for CICR. Vesicles released 30 nmol of Ca2+.mg" in response to 5 pM trigger Ca2+(-2.5 p~ free), although Ca2+release frequently followed a delay (asterisk) subsequent to addition of 5 p~ trigger Ca2+.The total amount of Ca2+released at low p~ ryanodine was unaltered. Preincubation of vesicles with elevated ryanodine (100 pM to 1 mM) resulted in a 65% loss of

+

ImM MgATP [Ryanodine] 1mM

I

m

SmM Crratine Phosphate

AA A

5 5 10

FIG. 2. Intraluminal calcium requirement for CICR. Dualwavelength spectroscopy of HSR Ca2+transport was performed as described in Fig. 1. HSR membranes (1 mg.ml-') were preincubated (1 min) at 25 "cin transport buffer containing 300 mM sucrose, 100 mMKC1, 20 mM PIPES (pH 7.0), 1 mM MgC12,5 mM CP, 12.5 units. ml" creatine phosphokinase and 40 ( A ) , 50 ( B ) , 60 (C), 70 (D), 80 ( E ) , 90 ( F ) , and 100 (C) FM Ca". Uptake was initiated by the addition of 1 mM Mg.ATP as indicated (arrows) and 10 PM Ca2+ pulses weremade to the sample cuvette (arrowheads). The entire experiment was reproduced three times at each level of Ca2+loading.

FIG. 3. Ryanodine effects upon HSR calcium uptake. Ca2+ transport was assayed spectroscopicallyas described in Fig. 1. Vesicles (1 mg.ml-') were preincubated for 5 min in the presence of 60 FM Ca2+ and various concentrations of ryanodine as indicated. Ca2+ uptake was initiated by addition of 1 mM Mg.ATP (solid arrow) to the cuvette and unless otherwise indicated 5 PM Ca2+pulses (arrowheads) were added to the cuvette during the steady state. Numbers below the arrowheads indicate the total final concentration of Ca2+ (PM) added to the cuvette at the point indicated. The traces were reproduced in three trials at each concentration of ryanodine.

Intraluminal Calcium and SR Calcium Release Ca2+ accumulation. The accumulated Ca2+ could then be released upon application of Ca2+ionophore (small arrow).At 1 p~ ryanodine, vesicles could accumulate the released Ca2+ and remained sensitive to subsequent Ca2+triggering of Ca2+ release. At ryanodine concentrations greater than 1 p ~ re, leased Ca2+was not accumulated in the presence of 5 mM initial CP. Further addition of 5 mM CP stimulated a slow Ca2+ reaccumulation. However, vesicles were insensitive to repeated Ca2+triggering of Ca2+release. Fig. 4 shows the effects of ryanodine addition upon Ca2+induced Ca2+release following complete uptake of medium Ca2+.As in Fig. 2, Ca2+pulses were added until release was observed. Increasing ryanodine resulted in a decrease in the intraluminal Ca2+threshold of Ca2+-inducedCa2+release. At 2 mM ryanodine, a delayed onset spontaneous Ca2+ release resulted without Ca2+triggering. The rates of initial Ca2+ release in the presence of ryanodine (953 nmol of Ca2+.mg HSR”) were not measurably different from controls (971 nmol of Ca2+.mg HSR”). However, the magnitude of Ca2+ triggered release increased with elevated ryanodine despite decreased vesicle Ca2+loading. As in Fig. 3, low ryanodine resulted in an initial fast Ca2+release followed by a slower release phase. At high ryanodine concentrations (100 p~ to 2 m M ) the initial fast phase was immediately followed by Ca2+ reaccumulation, the rate of which increased with elevated ryanodine. In several experiments, an additional Ca2+reuptake of 10-15 nmol of Ca2+.mg HSR” was observed during the subsequent 45 min (not shown). Fig. 5 shows that with CP supplementation prior to initiation of Ca2+ uptakethe released Ca2+, after addition of ryanodine (truce b ) , was reaccumulated with a much slower time

+

ImM Mg.ATP

+

20853

1mM MgATP

).

t t t t

t

t

t

t

t

t

t t t t t t FIG. 5. Ryanodine effects upon sustained CICR. In A , Ca2+ t t

t

t

uptake by HSR vesicles (1 mg.ml”) was monitored as described in Fig. 1 except for the presence of 20 mM CP. Vesicles were preincubated in the presence of 60 p~ Ca2+for 1min and uptake was initiated by the addition of 1 mM Mg.ATP to the cuvette (solid arrow). After accumulation of 60 nmol of Caz+.mg HSR-’, 10 p~ Ca2+additions were made to the cuvette to stimulate Ca2+release (small arrows). Repetitive Caz+releases were observed with sustained application of pulse Ca2+.In B , ryanodine (5 ptM) was added prior to partial stimulation of Ca2+release (long arrow). Each tracewas reproduced in four separate trials.

course than in the absence of ryanodine (trace a).In addition, ryanodine potentiated submaximal CICR. This is consistent with micromolar ryanodine stimulation of Ca2+channel opening (Fleischer et al., 1985). However, despite reaccumulation 1 mio of released Ca2+, subsequent Ca2+ releases were markedly inhibited and theuptake of added Ca2+also markedly slower. Thus refilling of the releasable pool of intraluminal Ca2+was also modified either directly or indirectly by ryanodine. [Ryanodine] In the control trace of Fig. 5, the magnitude of successive releases was diminished. This was accompanied by a reduction 2mM in the apparent open time of the channel. Contrary to the notion of an additional Ca2+ trigger site (see Nelson and Nelson, (1990)), Fig. 6 shows that the apparent loss of Ca2+ 400uM release correlated with a time-dependent formation of intraluminal Ca2+:Piprecipitates. Placement of a 5-min interval 8uM after the initial maximal Ca2+release (Fig. 6A, open arrow) and before the next addition of trigger Ca2+resulted in a much reduced subsequent Ca2+ release. The loss of Ca2+ Control release was identical to thatobserved after repetitive addition of trigger Ca2+to the cuvette during the same time interval. FIG. 4. Effect of ryanodine upon CICR. CaZ+ uptakeby HSR After another5-mininterval Ca2+ release waseffectively vesicles (1 mg.ml-’) was monitored as described in Fig. 1. Vesicles abolished. However, placement of the interval prior to subwere preincubated in the presence of 60 pM Ca2+,and Ca2+uptake was initiated by the addition of 1 mM Mg.ATP to the cuvette (solid maximal Ca2+release had little effect upon these and subsearrow). After accumulation of 60 nmol of Ca2+.mgHSR”, various quent releases (Fig. 6B).Therefore, diminution of Caz+release concentrations of ryanodine were added to thecuvette (open arrows). was related to time-dependent phenomena acting subsequent CICR was initiated by addition of 5 pM Caz+pulses (arrowheads) to to maximal CICR rather than due to intraluminal Ca2+conthe cuvette. At 400 PM ryanodine a minimum of 1-min preincubation tents, per se. In Fig. 6C, parallel filtration studies of Ca2+ with the drug wasallowed, although no ryanodine-induced Ca2+ uptake conducted under identical conditions showed that release was observed with much longer intervals (>lo min). At 8 p~ correlated, as expected, with the amount ryanodine, effects were independent of preincubation time and Ca2+ accumulated 45Ca2+ that was presented in pulses to the vesicles. Also observed pulses were added immediately following drug addition. The individual traces are representative of six separate trials conducted at each was the accumulation of Pi, which occurred biphasically with concentration of ryanodine. a steep rise of Pi accumulation observed after the accumula-

I

r?”.”.:

Intraluminal Calcium andS R Calcium Release

20854 A

I, t

tot

I

t

t

1 :2 200

0

100

200

300

400

500

Calcium Loaded (nmol/mg HSR)

FIG. 6. Intraluminal calcium and inorganic phosphate accumulation by HSR vesicles. In A and B, HSR Ca2+uptake was

value matches the intraluminal Ca2+ threshold for CICR determined in Fig. 2. Thus, inorganic phosphate appears to compete with an intraluminal Ca2+-binding compartment (presumably calsequestrin) for intraluminal Ca2+ aftermaximal CICR. The intraluminal Ca2+ sensitivity of ryanodine-induced Ca2+ release was then examined (Fig. 7). At defined Ca2+ loads, ryanodine was added in 5-pl aliquots at 30-s intervals until gradual upward excursions of the absorbance traces were observed during the lag period. The predictability of CICR under these assay conditions permitted Ca2+loading of vesicles with pulse ca'+ up to one 5 F M addition less than that required for CICR. The amount of ryanodine-induced Ca'' release was proportional to the intraluminal Ca'+ load. This was different from ryanodine augmentation of Ca2+-induced Ca2+release (see Fig. 4),which may reflect mixed effects of ryanodine-induced release and ryanodine stimulation of Ca2+induced release. The response of vesicles, partially loaded with Ca2+,to ryanodine (Fig. 7) contrasts with the response of Ca2+-depletedvesicles (Fig. 3). Vesicles were refractory to ryanodine-induced Ca2+release below 60 nmol. mg" intraluminal Ca", and, in all cases, release was followed by a slow reaccumulation of Ca2+. We were concerned that theelevated ryanodine concentrations required to elicit Ca'+ release may reflect possible drug modification and/or calculation errors during ryanodine purification and reconstitution. Fig. 8 shows that HSR vesicles bound ryanodine with both high (site A ) and low (site B ) affinity in the presence of millimolar nucleotide and submillimolar concentrations of Ca2+.The binding parameters obtained from Scatchard analysis were in agreement with earlier studies (Lai et al., 1989).

DISCUSSION assayed spectroscopicallyas described in Fig. 5. After maximal CICR was observed in A, 5-min intervals(open arrows) were placed between A recent study has shown that Ca2+-occupiedstates of subsequent additions of trigger Ca2+(10 p M total) to the cuvette to elicit Ca2+release. In B, the 5-min interval was placed prior to the calsequestrin within the lumen of SR cisternae are essential predicted observation of submaximal CICR by addition of 10 p~ ImM MgAW trigger Ca2+.Each trace was highly reproducible and representative of four trials. In C, HSR Ca2+transport was assayed radiometrically under similar conditions to that described above. Vesicles (1 mg. ml-') were preincubated with 60 PM total 45Ca2+,and uptake was initiated upon addition of 1 mM Mg.ATP in the presence of 300 mM sucrose, 100 mM KCI,20 mM PIPES (pH 7.0), 1 mMMgC12, 12.5 CALCIUM RYANODINE (nmol/mg) units.ml" creatine phosphokinase, 20 mM CP (total 2 ml volume). After 3 min, 3 X 50-pl aliquots were withdrawn and immediately A. 30 (90) filtered and rinsed with ice-cold buffer containing 300 mM sucrose, B. 20 (80) 100 mM KCl, 20 mM PIPES (pH 7.0), 100 p M EGTA, 5 mMMgC12, C. 10 (70) 873 50 p~ ruthenium red. The procedure was repeated after addition of D. 0 (60) 1454 D 120 nmol of"Caz+ to the remaining vesicles. The "Ca2+ addition/ vesicle filtration procedure was continued up to a total of six successive 120-nmol 45CaZ+ additions. The intravesicular retention of Ca2+ (open circles) was calculated by accounting for protein and volume changes resulting from aliquoting. The experimental protocol was U 1 min then replicated except for the addition of 'Oca2+.After rinsing, filters Ca2+ Ryanodine Lag Addition Addition Phase (13 mm HAWP-type) were placed into 600 pl of 6.7% (w/v) SDS and incubated overnight. Inorganic phosphate (filled circles) retained by FIG. 7. Effect of intraluminal calcium load upon ryanodinethe filter-trapped vesicles wasdetermined (see "Materials and Meth- induced calcium release. HSR vesicles (1mg.ml-') were allowed ods"). The datapoints in each experiment represent the mean of six to accumulate 60 nmol of Ca2+.mg protein-' upon addition of 1 mM observations from two sets of experiments (standard error less than Mg. ATP (solid arrow) as in Fig. 4.As indicated at thebottom of the 6%). trace, fixed amounts ofCa" were added as a series of 5 p M Ca2+ pulses to achieve a desired level of filling. This was followed after 30 s by addition of ryanodine in 5-pl aliquots (from 20 mM stock in HzO) tion of greater than 120 nmol of Ca2+.mg HSR". In addition, at 30-s intervals. Ryanodine addition was terminated upon observathe region afthe filter occupied by HSR was noticeably white tion of a definite progressive rise in the absorbance trace during the at elevated intraluminal Ca2+indicating formation of Ca2+:Pi lag phase. In the center of the traceare shown the amounts of precipitates. After accumulation of 500 nmol of Ca2+-mg ryanodine added to induce release at thecorresponding intraluminal HSR", intravesicular inorganic phosphatecontents were Ca2+load indicated within the brackets (nmol of Ca2'.mg HSR"). The Ca2+ release traces to the right are labeled according to the identical, suggesting a 1:l binding stoichiometry of Ca2+and loading scheme shown. The release traces from conditions B to D are phosphate. Back extrapolation of the steep portion of the superimposed upon the entire traceobtained under loading condition inorganic phosphate plot reveals a phosphate-free intralumi- A. The dataare representative of three entireexperiments conducted nal Ca'+ compartment of -100 nmol of Ca2+.mg HSR". This on different HSR preparations.

and S R Calcium Release

Intraluminal Calcium

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reports (Ohnishi, 1979, Nelson and Nelson, 1990). However, within a range of intraluminal Ca2+filling (60-100 nmol. mg”), release channel openingwas inversely dependent upon 1.25 the extraluminal trigger Ca2+ concentration. At these intra”50 luminal Ca2+ loads,the releasable Ca2+would be mostly bound (>go%) to calsequestrin (Ikemoto et al., 1989b). At intraluSITE A SITE B minal Ca2+ loads below 60 nmol. mg”, vesicles were comrk pletely refractory to CICR. Therefore, from Figs. 1 and 2 at least three different states of the channel (inactivated, activatable, and activated) can beidentified. Formation of these 11.8 118 states was dependent upon intraluminal Ca2+ and could be I I of Ca2+to actively expressed by manipulating the presentation accumulating vesicles. Thealkaloidryanodine was subsequently employed to probe the relationshipbetween Ca2+ channel states and intraluminal Ca2+ loading. With ryanodine presumably bound to 0 5 10 1525 20 30 35 40 the open channel of Ca2+-depleted HSR, during preincubation Bound (pmol/mg) (Fig. 3), subsequent ATP-dependent Ca2+ loading was proFIG. 8. Scatchard analysisof r3H]ryanodine binding to HSR gressively decreased with increased drug concentration. This from earlier studies (Fairhurst membranes. HSR vesicles (400 pg.ml”) were incubated in the is consistent with observations presence of 8 nM [3H]ryanodineand admixtures of cold ryanodine up andHasselbach, 1970; Fleischer et al., 1985),from which to 1mM final concentration (see “Materials and Methods”). At 1mM ryanodine was suggestedto “lock” the channelopen. Elevated [‘Hlryanodine total binding was 23.1 nmol.mg” HSR. Nonspecific ryanodine (1mM) did not result in apparent channel closure [“Hlryanodine binding was assumed to be a constant proportion of observed (Feher et al., 1988) total binding at each ryanodine concentration. The inset summarizes with increased Ca2+ retention as and affinity (&) at both high (Site A ) and was unaffected by prolonged (60-min) preincubation. binding site capacity (Bmax) and low (Site B ) affinity sites. Data are means of triplicate observaIkemoto et al. (1991) have recently shown that HSR Ca2+ tions in a single experiment and are representative of three inde- release is associated with a transient rise of intraluminal free pendent experiments conducted on different HSR preparations (less Ca2+. When ryanodinewas added to vesicles partially loaded than 5% standard error). with Ca2+ the alkaloid either ( a ) increased the intraluminal Ca2+ sensitivityof CICR or ( b ) directly induced spontaneous for theexpression of Ca2+- and caffeine-induced Ca2+ channel Ca2+release in a concentration-dependent manner that was opening and thus Ca2+ release (Ikemoto et al., 1989b). In this inversely proportional to intraluminal Ca2+load (Figs. 4 and study we havefurtherinvestigatedtheapparent coupling 6). Ryanodine-induced Ca2+ release was earlier observed by between the Ca2+ release channel and calsequestrin. We have Palade (1987) with Ca2+:Piloaded HSR vesicles. As observed demonstrated that expression of both activatory and inhibi- by Palade (1987), ryanodine-induced Ca2+release was always tory effects of ryanodine upon SR Ca2+release depends on preceded by a delay and a slow initial release of Ca2+.Of the t h e degree of intraluminalCa2+ loading. Furthermore, we known Ca2+-releasingagents,thisappearsto be a unique provide evidencethat the inhibitory effects of ryanodine upon property of ryanodine. The intraluminal Ca2+dependence of release may be associated with maintenance of the intraluryanodine stimulation of Ca2+ release is consistent with the minal Ca2+ binding compartment within a low affinity Ca2+ earlier proposal that Ca2+-induced conformational states of binding state. release channel opening (Ikemoto Observation of these effects was first approached by defin- calsequestrin regulate Ca2+ et al., 1989b). However, if the action of ryanodine ismediated ing conditions under which the channel could be indirectly solely through binding to the release channel, then Fig. 5 shown to be open or closed at different intraluminal Ca2+ suggests that conformational states of the release channel and loads. From observation of ruthenium red-inhibitable endogthe intraluminal-releasable Ca” compartment are coupled. enous Ca2+ release during vesicle preincubation (Fig. l), it can be concluded that the Ca2+ channel must have been open The dual action of ryanodine, to activate and then inhibit prior to addition of nucleotide. Upon addition of 1 mM Mg. Ca2+ release, could be explained if locking the channel in a ATP to thecuvette, an initial rapid accumulation of approx- particular state also prevented re-formation of high affinity by which imately 60 nmol of Ca2+-mg HSR”was observed. As shown Ca2+binding by calsequestrin.Themechanism elevated C P facilitates reaccumulation of extravesicular Ca2+ earlier (Gilchristet al., 1990), the rapid phasewas resolved by and hence demonstration of this effect of ryanodine may appropriate wavelength pair selection (675-790 nm) to eliminate Ca2+:ATP absorbance artifacts. The rate of initial Ca2+ simply be due to Ca2+:Piprecipitation. The experiments in uptake was indistinguishable from that observed in the pres- Fig. 6 demonstrating progressive loss of Ca2+release with the ence of 50 PM ruthenium red. The rapid phase was followed progressive accumulation of intraluminal inorganic phosphate by a secondary slow phase, which has been attributed to Ca2+(and thus Ca2+:phosphate precipitates) subsequent to maxichannel opening (Morii et al., 1985). Therefore, we assume mal CICR suggestthat thisis quite likely. However, inhibition t h a t initialrapiduptake of Ca2+must have accompanied of repetitive Ca2+ releasewas also observed with elevated ryanodine (not shown) where initial ryanodine-induced Ca2+ closure of a previously open channel. From Fig. 2 it is evident that the kinetic profile of HSR release was immediately followed by Ca2+ reaccumulation Ca2+ uptake,by KC1-washed vesicles, is dependent upon the (Figs. 4 and 7). This may reflect channel closure immediately experimentally manipulated distributionof Ca2+ betweenex- following Ca2+release under these conditions. On the other tra- and intraluminalCa2+pools. Regardless of the method of hand, elevated ryanodinedidnotstimulateapparentCa2+ vesicle Ca2+loading ( i e . Ca2+pulse versus single Ca2+ load), channel closure with Ca2+ depletedvesicles (Fig. 3). Clearly, maximal CICR (35 nmol of Ca2+.mg”) was observed after ryanodine effects are complex and are determined by ( a ) the accumulation of 100 nmol of Ca2+.mg HSR”. This intralu- intraluminal Ca2+load and ( b ) the activation state of the minal Ca2+ “threshold” for CICR is consistent with earlier release channel.

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Intraluminal Calcium and SR Calcium Release

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Ryanodine-induced Ca2+release and ryanodine activation of CICR may reflect the mechanism by which ryanodine has been shown to deplete the intraluminal Ca2+ stores in arterial smooth (Ashida et al., 1988; It0 et al., 1986; Iino et al., 1988) and cardiac muscle (Hunter et al., 1983; Hansford and Lakatta, 1987). In addition, the contrary inhibitory action of ryanodine upon Ca2+release may account for observation of ryanodine inhibition of Ca2+release (Marban andWeir, 1985; Fabiato, 1985), Ca2+oscillations (Lakatta et al., 1985; Cannell et al., 1985),and after contractions (Sutko and Kenyon, 1983) in cardiac cells. Depletion of intraluminal SR Ca2+pools and both ryanodine stimulationand inhibition of Ca2+ release may, therefore, represent different aspects of the same phenomenon. Observation of these seemingly different effects may then be determined by the degree of saturation of intraluminal Ca2+pools at the point of ryanodine administration. In thepresent study, absence of ryanodine stimulation of Ca2+ release below intraluminal Ca2+loads of 60 nmol. mg-' suggests that this pool is initially bound with high affinity to calsequestrin. Inhibition by ryanodine of intraluminal Ca2+ pool filling subsequent to release would indicate that ryanodine inhibitable conformational transitions of the release channel are reciprocally linked to transitions between high and low Ca2+affinity states of calsequestrin. greatly appreciate the assistance with ryanodine purification by Anthony Bore1 and Dr. Frank Abbott. Acknowledgments-We

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Ne-&;T. E., and Nelson, K. E. (1990)FEBS Lett. 263, 292-294 Nesterov, V. P. (1988)Fiziol Zh. (Kieu) 34, 60-66 Ohnishi, S. T. (1979)J. Biochem. (Tokyo)86, 1147-1150 Palade, P. (1987)J. Biol. Chem. 262,6149-6154 Pessah, I. N., Waterhouse, A. L., and Casida, J. E. (1985)Biochem. Bwphys. Res. Commun. 128,449-456 Raess, B. U., and Vincenzi, F. F. (1980)J. Phurmacol. Methods 4, 273-283 Rousseau, E., Smith, J. S., and Meissner,G. (1987)Am. J. Physiol. 253, C364C368

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