Adenine Nucleotide Stimulation of Ca2+-induced Ca2+ Release in Sarcoplasmic Reticulum*

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a ”Ca’+-release’’ and “control” fraction by differential and sucrose gradient centrifuga- tion. External Ca’+ (2-20 p ~ ) caused the release of 40 nmol of 4bCa’+/mg of protei& from Ca2+-release vesi- cles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca’+-in- duced CaZ+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca’+ concentration of 2 x M and Mg2+ concentration of 7 X M. “Ca2+ efflux from control vesicles was slightly inhibited at external Ca’+ concentrations that stimulated the rapid release of Ca’+ from Ca”-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca’+-induced Ca’+ release in media containing a “physiological” free Mg’+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP > CAMP - adenine > adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing “Ca2+ efflux from passively loaded Ca’+-release vesicles. La3+, ruthenium red, and procaine inhibited ca’+- induced Ca” release. Ca” flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca'+-release'' and "control" fraction by differential and sucrose gradient centrifugation. External Ca'+ (2-20 p~) caused the release of 40 nmol of 4bCa'+/mg of protei& from Ca2+-release vesicles passively loaded at pH 6.8 with an internal halfsaturation Ca2+ concentration of 10-20 mM. Ca'+-induced CaZ+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca'+ concentration of 2 x M and Mg2+ concentration of 7 X M. "Ca2+ efflux from control vesicles was slightly inhibited at external Ca'+ concentrations that stimulated the rapid release of Ca'+ from Ca"-release vesicles.
Adenine, adenosine, and derived nucleotides caused stimulation of Ca'+-induced Ca'+ release in media containing a "physiological" free Mg'+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP > CAMP -AMP -ADP > adenine > adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing "Ca2+ efflux from passively loaded Ca'+-release vesicles. La3+, ruthenium red, and procaine inhibited ca'+induced Ca" release. Ca" flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides.
Sarcoplasmic reticulum is a specialized intracellular membrane that regulates the contraction-relaxation cycle of skeletal muscle by releasing and reabsorbing Ca2+ (for reviews see Refs. [1][2][3][4][5][6][7][8]. Muscle contracts when myoplasmic Ca2+ reaches M, and relaxes as Ca2+ is pumped back into SR,' thereby establishing a new steady state at below M. Release of Ca2+ from SR is triggered by an action potential at the neuromuscular junction that is communicated to SR via the so-called T-system. The mechanism by which Ca2+ is released from SR remains to be clarified. A complicating factor has been that SR is inaccessible to direct electrical measurements using microelectrodes. More indirect approaches toward the study of Ca2+ release have made use of "skinned" muscle fibers or isolated SR vesicle fractions. In both systems, accumulated Ca2+ is released either by caffeine, ATP, an increase in external Ca2+, conditions that are thought to slow the Ca2+ pump of SR such as low Mg2+ or temperature, alteration in pH, or changes in the ionic environment that result in changes of surface membrane charge or a depolarized membrane (for reviews see Refs. 1, 2, 6, 8). On the basis of these observations, it has been proposed that physiological release of Ca2+ form SR is induced by Ca2+, "depolarization" of the SR membrane, changes in surface membrane charge, and/ or a pH gradient.
In this study, we have made use of previous observations of a differential Ca2+ release activity of muscle membrane fractions (9)(10)(11) to isolate from rabbit skeletal muscle by differential and sucrose gradient centrifugation a "Ca2+-release" fraction, which a t external Ca2+ of 2-20 FM rapidly released 45Ca2+. Mg' was inhibitory, whereas adenine nucleotides potentiated Ca2+-induced Ca2+ release. No or only little Ca2+ release activity was exhibited by another SR fraction which was utilized as a control ("control" fraction).

MATERIALS AND METHODS
Reagent~-'~Ca~+ was purchased from ICN Pharmaceuticals, Irvine, CA. Nucleotides including the ATP analog AMP-PCP were obtained from Sigma. All other chemicals were of reagent grade.
Isolation of Membranes-Sarcoplasmic reticulum membrane fractions derived from rabbit skeletal muscle were prepared as follows: 50 g of back and white leg muscle were minced and homogenized in 250 ml of 0.1 M NaC1, 5 mM Tris maleate, pH 6.8, at 4 "C for 60 s in a Waring blender. The homogenate was centrifuged for 30 min at 4,000 rpm (2,600 X g) in a GSA rotor in a Sorvall RC-2 centrifuge. Three crude membrane fractions were obtained from the supernatant by sequential centrifugation for 30 min at 10,000 X g, 30 min at 35,000 x g, and 60 min at 130,000 x g using a DuPont AH641 rotor. The pellets were resuspended in 0.3 M sucrose containing 0.1 M KC1 and 5 mM Tris/Mes buffer, pH 6.8, quickly frozen, and stored at -65 " C before use.
Isotope Flux Measurements-"Ca2+ efflux rates from vesicles passively loaded with rsCa2+ were determined by Millipore filtration. Unless otherwise indicated, vesicles were equilibrated for 30 min at 0 "C in a large volume (0.1-0.2 mg of protein/ml) of incubation medium (0.1 M KC1, 20 pM CaC12, and 10 mM K Pipes, pH 6.8), sedimented by centrifugation for 30 min at 100,000 X g, and resuspended in a small volume (5-10 mg of protein/ml) of incubation medium. Vesicles were passively loaded with 4'Ca2+ for 2 h at 22 "C in the presence of 5 mM %a2+ (0.2 mCi "Ca2+/ml). Vesicles were diluted 150-300-fold into an iso-osmolal, unlabeled release medium. 45Ca2+ efflux was terminated by placing 0.5-ml aliquots on 0.45-p HAWP Millipore filters followed by rapid rinsing with unlabeled medium. The time required to execute filtration and rinsing was about 20 s. An Update System 1000 Chemical Quench apparatus was used in experiments where rapid "Ca2+ efflux was inhibited at times of less than 5 s after vesicle dilution. One 10-ml glass syringe was filled with release medium and a second one with a quenching solution, and the sample (5 pl) was placed in a test tube. A first "push" delivered the release medium only. Rapid "Ca2+ release was stopped 1-3 s later by delivering with a second push an equal volume of release and quenching solution. The radioactivity retained on the filters was counted in 4.5 ml of a scintillation liquid which completely dissolved the filters (12).
Biochemical Assays-Protein was estimated by the method of Lowry et al. (13) using bovine serum albumin as a standard. Unless otherwise indicated, "basic" and MF-dependent, Ca2+-stimulated ATPase (Ca2+-ATPase) activities were determined at 32 "C. Basic ATPase activity was measured in 2 ml of a medium containing 10 mM K Hepes,pH 7.3,O.l M KC1,2.5 mM ATP, 6 mM M$+and 1 mM EGTA. Ca2+-ATPase activity was calculated by subtracting basic ATPase activity from the ATPase activity measured in the absence or presence of the ionophore A23187 (2 pg/ml) in 10 mM K Hepes, pH 7.3, 0.1 M KC1, 2.5 mM ATP, 5 mM M e , 100 PM Ca2+, and 100 ~L M EGTA. The reactions were stopped by the addition of 0.7 ml of 1.5 M HC10,. Inorganic phosphorus was determined on 1 ml of the protein-free supernatant (14) with elon as a reducing agent. The enzyme concentrations used resulted in less than 10% hydrolysis of ATP. Ca2+ loading rates were measured at 32 "C with a GCA/Mc-Pherson Spectrophotometer using the dye Antipyrylazo I11 for the determination of free Ca2+ as previously described (15). Ca2+ loading buffer solution contained 10 mM K Hepes, pH 7.3, 0.1 M KC1, 5 mM MgC12, 5 mM oxalate, 2.5 mM ATP, 100 p M ca2+, and 0.1 mM Antipyrylazo 111. Succinate cytochrome c reductase activity was estimated at 32 "C according to Fleischer and Fleischer (16).
Measurement of Free Mg?+ and Ca2+-Free M$+ concentration of nucleotide containing release media was determined with an Orion Divalent Cation Electrode. A standard curve was prepared by adding increasing amounts of M&12 to a buffer containing 0.1 M NaCl and 10 mM Na Pipes, pH 6.8. Free Ca2+ concentrations were calculated using for the Ca2+-EGTA complex an apparent binding constant of lo6 M" at pH 6.8 (17). Free Ca2+ concentrations of 5 p~ and greater were also directly measured with the use of the Ca2+-sensitive dye Antipyrylazo I11 (optical density = 0.3 at 651 nm) in a buffer containing 0.1 M KC1 and 10 mM K Pipes, pH 6.8. Changes in optical density at 651 nm were followed with a GCA/McPherson Spectrophotometer. Table I compares the Ca2+ release activities of rabbit skeletal muscle membrane fractions obtained by differential and sucrose gradient centrifugation. The muscle homogenate was subjectd to four sequential centrifugations of increasing speed to remove myofibrils and cell debris as well as to isolate three crude membrane fractions. After treatment with 0.6 M KCl, the crude fractions were further fractionated by sucrose gradient centrifugation.

Fractionation and Composition of Muscle Membrane Fractions-
Among the three crude membrane fractions, the 2,600- Ca2+ release properties of crude and sucrose gradient membrane fractions of rabbit skeletal muscle Crude membrane fractions obtained by differential pelleting and treated with 0.6 M KC1 were subfractionated into five membranous fractions of differing density on sucrose gradients (cf. "Materials and Methods"). Ca2+ release properties were determined as described in the legend to Fig. 1. Vesicles incubated for 2 h at 22 "C in the presence of 5 mM '%a2+ were diluted 300-fold into release media containing either 5 mM M$+ plus 1 mM EGTA or 50 p~ EGTA plus 50 PM Ca2+. The total amount of "Ca2+ trapped by the vesicles as well as the amount slowly released, were obtained by back extrapolation of the two release curves to the time of vesicle dilution (cf. Fig. 1). The per cent of Ca2+ release indicates the portion of trapped 4'Ca2+ that was rapidly released in the MF-free medium containing about 20 p~ free Ca2+. Data for the 2,600-10,000 X g crude and derived sucrose gradient fractions la-5a are the average of four preparations f S.E. Ca2+ release data of the remaining fractions are the average of three determinations of four pooled preparations. S.E. + 25% or less. 10,000 X g fraction displayed the highest Ca2+ release activity (Table I). About two-thirds of 45Ca2+ retained by the vesicles in a dilution medium containing 5 mM M e and 2 X lo-' M free Ca2+ was rapidly released on dilution into a medium containing 20 p~ free ca2+ (cf. Fig. 1). In the 10,000-35,000 x g fraction about half of the vesicles displayed Ca2+ release activity, whereas in the high speed 35,000-130,000 x g fraction only a small portion of the trapped 45Ca2+ was lost on dilution into the medium containing 20 p~ free Ca2+. On the sucrose gradients, in all three crude membrane fractions the highest Ca2+ release activity was recovered from the 35138% sucrose interface. For the 2,600-10,000 X g fraction, high Ca2+ release activity was also found in fractions sedimenting at the 321 35% and 29132% sucrose interfaces. Table I1 compares the enzymatic properties of two SR fractions displaying high or low Ca2+ release activity, and which are designated Ca2+-release fraction and control fraction, respectively. Both membrane fractions displayed properties characteristic of sarcoplasmic reticulum, i.e. accumulation of Caz+ in the presence of the Ca2+ precipitating agent oxalate, and Ca2+-stimulated, MP-dependent ATPase (Ca2+-ATPase) activity. Comparison of the levels of Ca2+-ATPase activity in the presence of the ionophore A23187 indicated that the Ca2+-ATPase content of the Ca2+ release fraction was about two-thirds of that of the control vesicles. Initial Ca2+ loading rates of the two SR fractions differed by a factor of 8. As shown below (Table IV), the low loading rate of the Ca2+-release fraction is likely due to the fact that an appre-  Table I1 were incubated for 2 h at 22 "C in a medium containing 10 mM K Pipes, pH 6.8, 0.1 M KCl, and 5 mM 45Ca2+. Vesicles were diluted at 22 "C 300-fold into unlabeled release media containing 10 mM K Pipes, pH 6 complex. In a second type of experiment, 45Ca2+ efflux from vesicles present in the two Me-free release media was slowed down by adding 1 volume of release medium and another one containing 10 mM K Pipes, pH 6.8, 0.1 M KC1, and amounts of M%+ and EGTA to yield final concentrations of 5 and 1 mM, respectively (c/. "Materials and Methods"). Aliquots of 0.5 ml were placed on Millipore filters and rinsed for 10 s with the final vesicle suspension medium, and amounts of radioactivity remaining with the vesicles on the filters were determined. Amounts of 45Ca2+ initially trapped by all of the vesicles (80 nmol/mg of protein for the Caz+-release fraction) as well as amounts not readily released from a subpopulation of vesicles (8 nmol/mg of protein for the Ca2+-release fraction) were obtained by back extrapolation to the time of vesicle dilution. In the inset in A , the time course of 45Ca2+ efflux from the vesicle population capable of rapid release was obtained by subtracting the amount of "Ca2+ not readily released (8 nmol/mg of protein).

TABLE I1
Enzymatic properties of vesicle /ructions displaying and lacking Caz+induced Ca2+ release activity Ca2+ release and control fractions correspond to sucrose gradient fractions (4a + 5a) and (3c + 4c) of Table I, respectively. "Ca2+ release indicates the portion of intravesicular "Ca2+ that is rapidly released by the vesicles in a medium containing 20 p M free Ca2+ (cf. Fig. 1). Enzymatic assays were carried out as described under "Materials and Methods." The data are the average of four determinations f S.E.

Ca2+ release Control fraction fraction
Rapid %a2+ release (%) 80 f 10 15 f 10 ATPase (pmol/mg protein/min) vesicles via a mechanism that is present in SR Ca2+-release vesicles but lacking in SR control vesicles.
Specific activity of basic ATPase, an enzyme associated with the plasmalemma and T-system of skeletal muscle (18), was low in both SR fractions. Since the surface membrane structures of rabbit skeletal muscle have been found in our laboratory to have a specific activity of about 5 pmollmg of protein/min (19), surface membrane content of the two fractions of Table I1 was estimated to be less than 5%. The Ca2+-release fraction was contaminated with inner mitochondrial membranes, as indicated by a succinate cytochrome c reductase activity of 0.15 pmollmg of protein/min. Inner mitochondrial membrane content of the control fraction was low.
45Ca2+ Efflux and Influx Rates of Ca2+-release Fraction-In Fig. 1, the Ca2+-release fraction of Table I1 was incubated in 5 mM 45Ca2+ for 2 h a t 22 "C. SR vesicles were diluted 300fold into an unlabeled release medium, were collected on Millipore filters a t time intervals ranging from 1/2 to 5 min, and the radioactivity remaining with the vesicles on the filters was determined. The amount of 45Ca2+ remaining with the vesicles depended on the composition of the release medium.
Vesicles diluted into a medium containing 5 mM M g + and 2 X lo-@ M free Ca2+ (1 mM EGTA and -20 p~ Ca2+) retained 80 nmol of Ca2'/mg of protein, that was slowly released with time (tIl2 = 5-10 min). In the presence of the ionophore A23187 (2 pglrnl), greater than 95% of 45Ca2+ was released within 30 s, indicating that the retained 45Ca2+ was trapped inside the vesicles (not shown). Omission of M$+ from the release medium containing 2 X lo-* free Ca2+ greatly accelerated the rate of 45Ca2+ efflux (tl/2 -30 s). A further dramatic increase in the initial 45Ca2' efflux rate was observed, as the free Ca2+ concentration of the Mg2+-free release medium was increased to 20 p~. By comparison, an increase in external Ca2+ from 2 X lo-@ M to 2 X M slightly decreased 45Ca2+ efflux from control vesicles (t1/2 -150 s uersus tlI2 -300 s) (Fig. 1B). Addition of 5 mM M e to the low Cap+ medium did not appreciably affect 45Ca2+ efflux. Fig. 1 are in accord with previous suggestions (1,2, 6) that sarcoplasmic reticulum contains a permeation system for Ca2+ which is activated by external Ca" and inhibited by M P . Release of greater than 90% of 45Ca2+ within 1-2 min suggested that a majority of the vesicles of the Ca2+-release fraction of Table I1 were capable of Ca2+-induced Ca2+ release. A small fraction of the vesicles seemed to lack this permeation system as indicated by slow release of Ca2+ that remained with the vesicles 1-2 min after dilution. Ca2+-release vesicles accounted for about half of the vesicles recovered in the 15 sucrose gradient fractions of Table I. M$+ and EGTA were found to effectively block further 45Ca2+ efflux when added to vesicles diluted into the Ca2+release medium containing 20 pM free Ca2+. Use of these two "quenching" agents therefore permitted us to stop Ca2+ release at time intervals ranging from 1-5 s (inset of Fig. 1A). A straight line was obtained from the Ca2+-permeable vesicle fraction suggesting that Ca2+ release followed first order kinetics. In the presence of 20 p~ free Ca2+, the vesicles released 45Ca2+ with an initial rate of about 20 nmol/mg of protein/s. 45Ca2+ influx was measured by varying the time of vesicle preincubation. Ca2+-release vesicles were preincubated from 3 min to 4 h at 22 "C in the presence of 5 mM 45Ca2+ before being diluted into the two media of Fig. 1 that blocked or promoted rapid Ca2+ release. Dilution into the Mg2+ medium that inhibited Ca2+ release showed a rapid increase in the amount of 45Ca2+ retained, indicating that initial 45Ca2+ inward movement was fast (Fig. 2). After a preincubation time of 2 to 4 h, 45Ca2+ had nearly equilibrated across the vesicle membranes. Vesicles were therefore routinely preincubated for 2 h at 22 "C prior to the Ca2+-release studies. The ability of the vesicles to release most of their "Ca2+ within 10 s in the Ca2+release promoting medium was independent of the time of preincubation, suggesting that Ca2+ release was not altered by preincubation for 2 h at 22 "C. Isolation of the membrane fractions or preincubation of the vesicles in the presence of the potent serine protease inhibitor, diisopropyl fluorophosphate at a concentration of 1.5 mM, also showed no effect (not shown).

Data of
Coupling of Ca2+ Movement to Other Zons-The presence of an obligate Ca2+:Ca2+ exchange reaction was assessed by di-  Table I1 were incubated at 22 "C for the indicated time in 10 mM K Pipes, pH 6.8,O.l M KC1, and 5 mM 46ca2'. Vesicles were then diluted 300-fold into media containing 10 mM K Pipes, pH 6.8, 0.1 M KC1, and 5 mM M e plus 1 mM EGTA (O), or 50 @M EGTA plus 50 PM Ca2+ (0). At 10 s after the initial vesicle dilution, 46Ca2+ efflux from vesicles present in the M$+-free release medium was inhibited by adding an equal volume of medium containing 10 mM K Pipes, pH 6.8, 0.1 M KC1, 10 mM M$+, and 2 mM EGTA. The amount of 45Ca2+ remaining with the vesicles was obtained by back extrapolation to the time of vesicle dilution (0) or addition of the quenching medium (0) (cf. Fig.1). The difference between the upper and lower curves indicates the amounts of 45Caz+ released within 10 s in the M e -f r e e release medium (0). TIME ihr) luting 45Ca2+ loaded vesicles into release media containing 50 PM unlabeled Ca2+ or 50 p~ 45Ca2' with a radioisotope specificity identical with the one used in the incubation medium. Ca" release was stopped after 5 and 10 s by the addition of an equal volume of buffer containing 10 mM M P and 2 mM EGTA (cf. Fig. 1). An identical rapid 45Ca2+ efflux rate was observed in both release media suggesting that outward movement of 45Ca2+ was not coupled to inward movement of Ca2+. In agreement with this suggestion, vesicles loaded with 5 mM unlabeled Ca2+ and then diluted into a release medium containing 45Ca2+ did not accumulate the radioisotope. Ca2+ release also appeared to be not directly coupled to any specific monovalent cation since rapid release was observed in KC1, NaC1, or choline C1 media. Native SR vesicles are permeable to H+ (ZO), so it was not possible to determine whether Ca2+ efflux was coupled to H+.
Addition of 5 WM valinomycin to the KC1 release medium did not appreciably affect 45Ca2+ release, suggesting that Ca2+ efflux did not result in the formation of a large membrane potential, which in turn might have limited the extent of Ca2+ release. Probably, the intrinsic permeability of the SR vesicles to K+, C1-, and H+ (7, 8) was sufficiently high to dissipate any potential that might have formed during electrogenic Ca2+ efflux.
Effect of External Ca2+ and Mg2+ on 45Ca2+ Release-The effect of varying external Ca2' and Mg2+ concentrations on the initial Ca2+ efflux rate from SR vesicles capable of rapid Ca2+ release and equilibrated with 5 mM 45Ca2+ is shown in Fig. 3. A maximal initial Ca2+ efflux rate of 18 nmol of Ca2+/ mg of protein/s was observed in Me-free media containing between 2 and 20 PM free Ca2+. Both a decrease in free Ca2' concentration to 2 X M or an increase to M decreased the initial Ca2+ release rate to less than 2 nmol/mg of protein/ s. Addition of M e to the release medium slowed down 45Ca2+ efflux. The initial Ca2+ release rate was approximately halfmaximal at 7 X M MgZ+ in a medium containing 20 PM free Ca2+. The intracellular concentration of free MgZ+ has been estimated to be 0.6 mM in frog skeletal muscle (21). Fig.  3 shows that Ca2+-induced Ca2+ release was nearly fully abol-  Table  I1 was incubated for 2 h at 22 "C in a medium containing 20 mM K Pipes, pH 6 ished at this physiological concentration of 0.6 mM M F . La3+, ruthenium red, and procaine have been reported to inhibit Ca2+-induced Ca2+ release from SR (1,2,6,(22)(23)(24). At a concentration of 10 p~, La3+ and ruthenium red reduced the initial Ca2+ release rate to less than 0.5 nmol/mg of protein/s. Procaine at a concentration of 10 mM lowered the initial Ca2+ release rate from 18 to about 5 nmol/mg of protein/s (not shown). Effect of Nucleotides on 45Ca2+ Efflux-Inhibition of Ca2+induced Ca2+ release by physiological concentrations of M F raised the possibility that a regulatory factor might have been removed during purification resulting in increased M e -s e nsitivity of the Caz+-release system of sarcoplasmic reticulum. Data of Fig. 4 and Table I11 suggest that adenine nucleotides may play such a role. Ca2+-release vesicles equilibrated with 5 mM 45Ca2+ rapidly released all of their Ca2+ when diluted into a medium containing about 10 p~ free Ca2+, 1.5 mM M$+, and 1 mM of the nonhydrolyzable ATP analog, AMP-PCP. The free M F concentration of the medium determined with an ion electrode was 0.7 & 0.1 mM. ca2+ releasing action of AMP-PCP was also observed when vesicles were first diluted into a medium containing 1.5 mM Mg2+ and 10 p M free Ca2+ followed by the addition of the nucleotide to a final concentration of 1 mM. The stimulatory effect of AMP-PCP was less pronounced when the free Ca2+ concentration was lowered to about IO-' M by the addition of an excess of EGTA (Table 111). An increase in M F concentration to 10 mM nullified the stimulatory effect of AMP-PCP. Vesicles did not actively take up 45Ca2+ when incubated in a medium containing 1.5 mM M$+, 1 mM AMP-PCP, and 20 p~ free Ca", suggesting that the nucleotide did not exert its effect by activating the Ca'+-transport system of sarcoplasmic reticulum. In another control experiment, we found that AMP-PCP did not significantly increase 45Ca2+ efflux from control vesicles.
Other nucleotide triphosphates did not significantly affect the Ca2+-release system of sarcoplasmic reticulum. No increase in the release rate was seen when GTP, ITP or UTP, or the two nucleotide analogs GMP-PCP or UMP-PCP were added at a concentration of 1 D M to media containing 1.5 mM M$+ and 10 FM free Ca2+ (Table 111) Table 11, preincubated for 2 h at 22 "C in the presence of 5 mM 45Ca2+, was diluted into release media containing 20 mM K Pipes, pH 6.8, 0.1 M KC1, approximately lo-' M free ca2+ (1 mM EGTA and ca2+ at a final concentration of 12.5 pM) or lo-' M free Ca2+ (50 UM EGTA and Ca2+ at a final concentration of 55 PM), the indicated amounts of M%+, and nucleotides at a concentration of 1 mM. Initial "Can+ efflux rates were determined by adding at 5, 10, and 30 s (in the case of AMP-PCP at 1, 2, and 3 s ) after vesicle dilution a quenching solution containing amounts of M%+ and EGTA to yield final concentrations of 6 and 2 mM, respectively (cf. Fig. 1 adenine and, to a lesser extent adenosine behaved like AMP-PCP in that they accelerated Ca2+ release in media containing about 0.6 mM free Mg2+. By contrast, when caffeine, reported to augment Ca2+-induced Ca2+ release (1, 2, 6), was added to a medium containing 0.6 mM Mg2+, 45Ca2+ efflux was only minimally stimulated. GMP at a concentration of 5 mM accelerated 45Caz+ efflux 1.5-fold, suggesting that other nucleotides may activate the Ca2+-release system of SR when present at a high concentration. Both AMP-PCP and ADP exerted their effect on the Ca2+release system of SR in a dose-dependent manner, with AMP-PCP being the more effective of the two nucleotides (Fig. 5).
In a medium containing 1 mM AMP-PCP, vesicles released about 90% of the rapidly releasable 45Ca2+ at 5 s postdilution. Taken together, the data of Table 111 and Figs. 4 and 5 suggest that the Caz+-release system of SR may be regulated by nucleotides. Adenosine nucleotides seem to be the preferred substrate since the ATP analog AMP-PCP was the only one of the various nucleoside triphosphates tested that significantly activated Ca2+ release. Dependence of 45Ca2+ Efflux on Internal Ca2+ Concentration and pH-A vesicle fraction shown to release rapidly Ca2+ was incubated for 2 h at 22 "C in media containing trace amounts of 45Ca2+ and between 2 and 62 mM Ca2+. As the Ca2+ concentration of the incubation medium increased, vesicles retained increasing amounts of Ca2+ upon dilution into a medium containing 6 mM Mg2+ and low concentrations of free Ca2+ (Fig. 6). 45Ca2+ efflux was slow in media containing 0.6 mM M%+ and between 5 and 10 p~ free Ca2+. In accordance with data of Fig. 1 and Table 111, omission of M$+ or addition of AMP-PCP caused a dramatic increase in the initial 45Ca2+ efflux rates. A half-maximal initial release rate of about 100 nmol of Ca2+/mg of protein/s was obtained for vesicles filled with a Ca2+ concentration of 10-20 mM and subsequently diluted into the nucleotide containing medium. Fig. 7 illustrates that Ca2+-induced Ca2+ release is influenced by pH. Vesicles were preincubated over a pH range of 6.0-7.6 and then diluted into media of the same pH. Dilution of vesicles into media containing 5 mM MgZ' and 1 mM EGTA resulted in retention of 50-75 nmol of Ca2+/mg of protein, which was slowly released with time (tlI2 -5 min), indicating that the vesicles could form a permeability barrier for Ca2+ over the pH range studied. The amount of 45Ca2+ released 5 s postdilution moderately increased as the pH of the 0.6 mM M e medium containing micromolar concentrations of free Ca2+ was raised from pH 6.0 to 7.6. By contrast, 4sCa2+ efflux greatly increased when the pH was raised from 6.0 to 6.8 in the two Ca2+-release promoting media. At pH 7.2, the vesicles had essentially lost all of their rapidly releasable 45Ca2+ within 5 s when diluted into media containing either micromolar concentrations of free Ca2+ or, in addition, 1.5 mM MgZ' plus 1 mM AMP-PCP. At pH 6.3, a 5-10-fold increase of free Ca2+ concentration in the MgZ'-and nucleotide-free release medium did not increase 45Ca2+ efflux, suggesting that H+ did not inhibit Ca2+ release by simply lowering the affinity of an activating cation binding site for Ca2+ (not shown).
Ca2+ Release from Actively Loaded Vesicles--In contrast to the experiments described above where SR vesicles were passively loaded with 45Ca2+, the data of Table IV was Table 11) was inhibited by adding Ng, (an inhibitor of the electron transport chain) as well as the H+carrier carbonyl cyanide p-trifluoromethoxyphenylhydrazone (a potent uncoupler of oxidative phosphorylation in mitochondria). Actively loaded vesicles were placed on a Millipore filter and rinsed for 10 s with washing solutions 3f different composition. Control vesicles retained similar amounts of  Ca'+-release and control fractions (60-70 pg of protein) of Table gradient inhibits further ATP hydrolysis SO that in vesicles I1 were incubated for 5 min at 22 "C in 1 ml of 20 mM K Pipes, pH incapable of Ca2+ release, an ATP hydrolysis rate of 0 would 6.8, 0.1 M KC19 5 NaN39 0.8 pM cyanide Pbe expected to be approached. Under steady state conditions trifluoromethoxyphenylhydmone, 100 PM EGTA, 100 pM &Ca'+, and in the presence of about 4 mM free M$+, Ca2+-ATpase activity either 5 or 0.75 mM M P . %a2+ uptake was initiated by the addition on a 0.45-p Millipore filter and rinsed for 10 s with a medium mg of Proteinbin Or 6-7 nmol/mg of Prokin/s at 22 "c containing 20 mM K Pipes, pH 6.8, 0.1 M KC1, and the indicated (Table IV). Relatively high ATPase activity of the vesicles concentrations of M$+, EGTA, Ca'+, AMP-PCP, and adenine. b-was a puzzling observation considering that the passive Ca2+ dioactivity retained by the vesicles on the filters was determined. permeability of both types of vesicles under similar conditions Ca'+-ATPase activities were determined as described under "Mate-was quite low. AT^^^^ activity of 6-7 nmol/mg of rials and Methods" except that the assay temperature was lowered to 22 oc and the composition of the assay medium was changed to 20 s would correspond to a theoretical Ca2+ net efflux rate of 12-  rier for Ca2+ in high or low M$+ media. Taken together, Ca2+ flux and ATP hydrolysis data of Table IV support our contention that a subpopulation of sarcoplasmic reticulum vesi-100 p~ ca'+, 100 p~ EGTA 18 : i cles contains a Ca2+-permeation system that is controlled by Ca2+, Mp", and ATP.  (Table IV). By contrast, the amount of 45Ca2+ retained by the Ca2+-release fraction depended on the composition of the two media. Maximal amounts of %a2+ were retained when vesicles were loaded and washed in media that were shown above to block Ca2+ release from passively loaded vesicles, i e . media containing a free M$+ concentration in an excess of 4 mM. Omission of M e from the Ca2+-free rinsing medium caused release of 25% of the sequestered %a2+. Rinsing the vesicles with media containing a free Ca2+ concentration of about 16 PM, or containing in addition to 0.6 mM free M$+, the ATP analog AMP-PCP or adenine resulted in rapid efflux of 50-70% of the sequestered 45Ca2+ from the vesicles. Ca2+-release vesicles actively loaded in a medium with a low free M$+ concentration retained only low amounts of 45Ca2+, regardless whether vesicles were rinsed with a washing medium promoting or blocking Ca2+ release. Since control vesicles accumulated high levels of 45Ca2+ in the low Mg2+ incubation medium, it appeared that low retention of 45Ca2+ in the Ca2+-release fraction was not due to a suboptimally operating Ca2+ pump, but rather, to rapid outflow of the transported Ca2+ via the Ca2+-release system present in these vesicles.

DISCUSSION
This study has shown that rabbit skeletal muscle homogenates contain sarcoplasmic reticulum vesicles which differ in their permeability to Ca2+. Ca2+-release vesicles, which accounted for about half of the isolated SR vesicles, appear to contain a Ca2+-permeation system that allows the rapid efflux of Ca2+. The Ca2+ efflux rate is regulated by external Ca2+, Mg+, and adenine nucleotides. Control membrane vesicles, by definition, seem to lack the permeation system that mediates rapid Ca2+ release under the experimental conditions of this study. Absence of the Ca2+-release system from a population of SR vesicles suggests that rapid Ca2+ efflux required a membrane component specific to Ca2+-release vesicles.
Longitudinal sections and terminal cisternae of sarcoplasmic reticulum have been distinguished in electron micrographs of skeletal muscle (25). A structure known as a triad is formed where the terminal cisternae of SR abuts on invaginations of the surface, the transverse tubule (T-system). Isolation of vesicle fractions enriched in the different segments of the SR structure has been described (26)(27)(28). Vesicle fractions displaying a high Ca2+-release activity (cf . Table I) exhibited a sedimentation behavior similar to that previously observed for "heavy" free terminal cisternae vesicles (26) as well as vesicles attached to transverse tubule in the form of diads or triads (27,28). Control membranes were preferentially recovered from the low density region of the sucrose gradients, similar to "light" SR vesicles (26) derived from the longitudinal sections of SR.
Ca2+ is thought to be released from the terminal cisternae of SR in response to a T-system action potential (29,30). It would be expected then that disruption of SR during homog-enization might result in the formation of some vesicles that contain, and others which lack, the Ca2+-release system. Weber and Herz (9) first reported a differential Ca2+ release sensitivity of muscle membrane fractions. Caffeine released 25-50% of the accumulated Ca2+ when a 2,000-8,oOO x g membrane fraction was used, whereas high speed fractions released no more than 12%. In two recent studies, the majority of vesicles obtained by centrifugation between 2,400 and 8,600 x g (10) or 4,000 and 10,000 x g (11) possessed a "Ca2+-gated" cation channel, whereas only a small portion of the vesicles sedimenting at higher speeds exhibited Ca2+-induced Ca2+ release activity. Miyamoto and Racker (23) observed Ca2+induced and membrane potential-dependent Ca2+ release from actively loaded, terminal cisternae fractions, but not from light SR vesicle fractions. In addition in some (31, 32) but not all (12, 33) studies, alterations of surface charge distribution or transmembrane potential, induced by a change in ionic composition, have been reported to cause Ca2+ release from terminal cisternae and triad-enriched vesicle fractions.
One popular hypothesis of Ca2+ release from SR in vivo is the Ca2+-induced Ca2+ release or Ca2+-triggering hypothesis which states that small amounts of Ca2+ entering the sarcoplasm during an action potential induce release of additional amounts of Ca2+ sufficient to activate muscle contraction (1,2,6). Two proteins generally considered to be capable of mediating rapid Ca2+ release from SR are the Ca2+ transport  (44), showed no effect (43), or as observed in this study, dramatically increased (41) the initial 45Ca2+ efflux rate. Further increase of external ca2+ to 10-3-10-2 M was without effect in one study (43), but otherwise reduced 45Ca2+ efflux to low values. Using a light-scattering method, Yama-mot0 and Kasai (24) found that choline influx into SR vesicles via the Ca2+-release channel is controlled by Ca2+ and M F . Measurement of choline influx rates indicated an apparent activation constant of 3 X M for Ca2+ and apparent inhibition constants of 1.4 X M for Mg2+ and 2.2 X M for Ca2+. In this study, 45Ca2+ efflux was half-maximally stimulated at 6 X M 45Ca2+, indicating an activating site with a high affinity to Ca2+ (see Fig. 3). The inhibiting site(s) had an apparent lower affinity, 45Ca2+ efflux being half-maximally inhibited at 7 X M M$+ and 2 X M Ca2+. An argument against the Ca2+-triggering hypothesis has been that Ca2+-induced Ca2+ release from skinned muscle fibers and SR vesicles is inhibited by physiological concentration of M$+ (1, 2, 6). Additional parameters have therefore been considered to control rapid release of Ca2+ from SR such a change in transmembrane potential (23,45), surface charge (461, or pH (47). This study shows that adenine nucleotides dramatically stimulate Ca2+ release from passively and actively loaded SR vesicles. Involvement of nucleotides in the Ca2+ release process has been previously reported. At a very low level of free M e , ATP stimulated the release of Ca2+ in skinned muscle fibers (48). Ogawa and Ebashi (37) found that AMP-PCP induced the release of Ca2+ from actively loaded vesicles (37 nmol/mg of protein/30 s). Chiesi and Wen (35) observed that the rapid phase of ATP-induced Ca2+ release from vesicles passively loaded with 10 mM Ca2+ was composed of two components, one involving the phosphorylated intermediate of the Ca2+-ATPase and the other also being induced by AMP-PCP (10 nmol of Ca2+/mg of protein/5 s), and therefore apparently requiring only binding of the nucleotide to the catalytic sites of the enzyme. Whether rapid release of Ca2+ was mediated by binding of the nucleotide to the Ca2+-ATPase or to another component of SR is not clear from the data presented in the previous studies. We observed that Ca2+ releasing action of AMP-PCP paralleled that of Ca2+-induced Ca2+ release in that similar amounts of Ca2+ were rapidly released from passively and actively loaded vesicles in media containing micromolar concentrations of free Ca2+ and no or 1.5 mM M$+ plus 1 mM AMP-PCP. The ability of AMP-PCP to stimulate 45Ca2+ efflux from vesicles capable of Ca2+-induced Ca2+ release, but not from vesicles that lack this mechanism but nevertheless contain the Ca2+-ATPase, suggests that AMP-PCP exerted its effect through a structure different from that of the Ca2+-ATPase. Since both types of vesicles contained the Ca2+-ATPase, it could not be excluded, however, that the Ca2+-ATPase in conjunction with another component formed the Ca2+ channel of the Ca2+-release vesicles. In addition, it may be noted that we could not rule out the possibility that rapid Ca2+ release observed in the presence or absence of adenine nucleotides is mediated by two separate rather than a single pathway.
During the review of the manuscript we became aware that Morii and Tonomura (49) had also found that various adenine nucletoides (ATP, AMP-PNP, ADP, AMP) accelerated the release of Ca2+ from passively loaded veiscles. Other trinucleotides (CTP, GTP, ITP, UTP) and caffeine had no effect on the release of Ca2+. Rather one of these (CTP) inhibited the releasing effect of AMP-PNP. In addition and in disagreement with the present study, CAMP and adenosine were found to be ineffective. The degrees of effectiveness of some of the nucleotides may be due to differences in the assay conditions used by us and Morii and Tonomura (49). Their measurements of Ca2+ release a t 0 "C in media containing 5 mM M$+ resulted in relatively low Ca2+ release rates of 1-5 nmol/mg of protein/s. Kinetic studies of the Ca2+-releasing action of the adenine nucleotides indicated that Ca2+ efflux was activated by AMP with a Hill coefficient of 1 and an apparent dissociation constant of 2 mM (49). In the presence of 5 mM M$+, the addition of 1 mM ATP or 1 mM AMP increased the amount of Ca2+ released from 0 to 60%, as the free Ca2+ concentration was increased from 0.06 to 0.24 pM. Morii and Tonomura proposed that the Ca2+-release channel of SR was activated by the binding of one molecule of adenine nucleotide. Different external Ca2+ concentrations were thought to be required to open a heterogeneous population of activated channels in an all or none fashion. We observed that both in the absence and presence of 1.5 mM M F plus 1 mM AMP-PCP, the rate but not the total amount of Ca2+ release depended on the external Ca2+ concentration. This observation would seem to favor a model in which all channels are capable of opening, however, where frequency or duration of channel opening or the rate of ion movement through the channel are regulated by external Ca", M$+, and adenine nucleotide. The role of M e in the Ca2+ release process was not considered by Morii and Tonomura (49).
The Ca2+ releasing action of adenine is of interest, since caffeine, a related compound, is known to cause Caz+ release from skinned frog skeletal muscle and SR vesicles (1,2,6). In addition to external activating and inhibiting divalent cation sites, it has been suggested that the Ca2+-release channel of SR possesses an activating receptor site for caffeine (24,50). Mammalian skeletal muscle is less sensitive to caffeine than frog skeletal muscle (10,37), which may explain the relative ineffectiveness of caffeine in stimulating 45Ca2+ release in our studies.
Ca2+-and nucleotide-induced Ca2+ release approached saturating values of about 80 and 200 nmol of Ca2+/mg of protein/s, respectively, at high internal Ca2+ concentration indicating that rapid 45Ca2+ release was preceded by binding of Ca2+ to an internal low affinity site (& = 10-20 mM). At a "physiological" internal free ca2+ concentration of 5 mM (7), 45Ca2+ efflux rate of -40 nmol/mg of protein/s was observed in a medium containing AMP-PCP. While this efflux rate was 10-fold and more higher than those observed in most previous studies, it seems to be nevertheless low when compared with 45Ca2+ release rates in vivo. In fast twitch muscle fibers, the release of about 0.1-0.2 pmol of CaZ+/g of muscle in 5 ms is required to initiate muscle activity (51). This corresponds to a release rate of 4-8 pmollmg of protein/s, using an SR content of 5 mg of protein/g of muscle (52) and assuming that Ca2+ release occurs over the whole reticulum structure. Following the suggestion that Ca2+ release is limited to the terminal cisternae region of SR (29,30) and that it accounts for one-third of the reticulum structure (53), a release rate of 12-24 pmol of Ca2+/mg of protein/s is estimated. Thus, maximal release rates observed in this study for vesicles passively loaded with 5 mM Ca2+ would correspond to less than 1% of the in vivo rate. A release rate of 40 nmol of Ca2+/mg of protein/s corresponds to an ion flux rate of about lo4 Ca2+/channel/s, assuming that there is on an average only one channel/vesicle, an average vesicle diameter of 0.1 pm (26) and an intravesicular volume of 2 pl/mg of protein (54). Since the turnover of an ion channel with a half-saturation concentration of 20 mM can be as high as lo7 s-' (55), it may be that more physiological assay conditions such as an increase of nucleotide concentration from 1 to 5 mM, temperature from 22 to 37 "C, and pH from 6.8 to 7 or 7.2 significantly increases in vitro Ca2+-release rates. However, to measure such rapid rates, it will be necessary to carry out the release experiments on a shorter time scale than has been possible in the present study.
In media containing ATP and a relatively low concentration of free Mg2+ and Ca2+ at a concentration of 10-6-10-5 M, Ca2+release vesicles rapidly released their Ca2+, resulting in a low steady state level of Ca2+ uptake during active transport (Refs. 10 and 23; Table IV). This observation raises an important question: how is Ca2+ release interrupted once the myoplasmic Ca2+ concentration has been raised to about 10-6-10-5 M during the event of excitation-contraction coupling? One possibility would be that under intracellular conditions, the Ca2+transport system of SR can sufficiently remove Ca2+ near release sites to cause a rapid inactivation of the Ca2+-release channels. Alternatively, it is conceivable that a protein responsible for terminating Ca2+ release from SR or another regulatory factor has been removed during vesicle isolation. In this regard it is of interest that exposure of the membrane fractions to salt results in dissociation of SR and T-system membranes as well as loss of "feet" proteins (19,40,56) suggesting that perhaps a component of the triad missing from the Ca2+-release vesicles mediates the termination of 45Ca2+ release.