ARCHIVES

OF BIOCHEMISTRY

AND

Vol. 292, No. 2, February

BIOPHYSICS

1, pp. 512-521,1992

Photooxidation of Skeletal Muscle Sarcoplasmic Reticulum Induces Rapid Calcium Release’ Janice

Stuart,*‘2

Isaac N. Pessah,?

Terence

G. Favero$

and Jonathan

J. Abramson$,3

*Departments of Chemistry and $Physics, Environmental Sciences and Resources Program, Portland State University, Oregon 97207; and TDepartment of Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis, California 95616

Portland,

Received July 24, 1991, and in revised form October 3, 1991

The photooxidizing xanthene dye rose bengal is shown muscle sarto induce rapid Ca2+ release from skeletal coplasmic reticulum (SR) vesicles. In the presence of light, nanomolar concentrations of rose bengal increase the Ca2+ permeability of the SR and stimulate the production of singlet oxygen (‘0,). In the absence of light, no ‘Oz production is measured. Under these conditions, higher concentrations of rose bengal (micromolar) are required to stimulate Ca” release. Furthermore, removal of oxygen from the release medium results in marked inhibition of the light-dependent reaction rate. Rose bengal-induced Ca2+ release is relatively insensitive to Mg2+. At nanomolar concentrations, rose bengal inhibits [3H]ryanodine binding to its receptor. j3,y-Methyleneadenosine 5’-triphosphate, a nonhydrolyzable analog of ATP, inhibits rose bengal-induced Ca2+ release and prevents rose bengal inhibition of [3H]ryanodine binding. Ethoxyformic anhydride, a histidine modifying reagent, at millimolar concentrations induces Ca2+ release from SR vesicles in a manner similar to that of rose bengal. The molecular mechanism underlying rose bengal modification of the Ca2+ release system of the SR appears to involve a modification of a histidyl residue associated with the Ca2+ release protein from SR. The light-dependent reaction appears to be mediated by singlet oxygen. 0 1992

Academic

Press,

Inc.

1 This work was supported by grants from the American Heart Association (87.915), the American Cancer Society (CH-445), and NIH (lR15GM44337-01) to J.J.A.; by an NIH Postdoctoral Fellowship (HL08388) to T.G.F.; and by NIH (ES05002) to I.N.P. This is Portland State University Environmental Sciences and Resources Publication 263. * Present address: Department of Pharmacology, Oregon Health Sciences University, Portland, OR 97201. 3 To whom correspondence should be addressed at Department of Physics, Portland State University, P.O. Box 751, Portland, OR 97207. 512

The sarcoplasmic reticulum is an internal membrane system within muscle cells which is responsible for controlling the myoplasmic Ca2+ concentration and thereby regulating contraction and relaxation of skeletal muscle (1). Myoplasmic Ca2+ is actively accumulated into the SR4 by Ca’+, Mg2+-ATPase, which results in muscle relaxation. The molecular mechanism underlying the Ca2+ release process from SR is less well understood. Chemical modification of the SR has been reported to cause release of Ca2’ from SR vesicles. Oxidation of reactive thiols induces rapid Ca2+ release from SR (4-9). Oxidation-induced Ca2+ release is stimulated by adenine nucleotides (5, 6, lo), and is inhibited by ruthenium red (4-6), local anesthetics (4), and Mg2+ (4-8). Modification of amino groups has also been shown to stimulate Ca2+ release from passively loaded SR vesicles (11, 12). Modification of 1 mol lysine and l-2 mol histidine per mole of ATPase has been reported to inhibit Ca2+, Mg2+ATPase activity (13). Also, photooxidation of SR proteins results in inhibition of Ca2+-ATPase activity and of active Ca2+ uptake (14-B). This oxidation involves either histidy1 (14-16) or tryptophanyl residues (16). Nanomolar concentrations of ryanodine, a neutral plant alkaloid, has been shown to bind in a Ca2+-dependent manner to specific sites localized at the triadic junction (19). It has also been shown to modify the Ca2+ permeability of the SR (20), and to bind directly to the Ca2+ release protein (21). Ryanodine binding is stimulated by ’ Abbreviations used: SR, sarcoplasmic reticulum; Hepes, 4-(2-hydroxyethyl))1-piperazineethanesulfonic acid; ATP, adenosine 5’.triphosphate; AcPO,, acetyl phosphate; AMP-PCP, /3,-r-methyleneadenosine 5’-triphosphate; ASIII, arsenazo III; APIII, antipyrylazo III; EFA, ethoxyformic anhydride; RNO, p-nitrosodimethylaniline; NADH, nicotinamide adenine dinucleotide, reduced form; NAD, nicotinamide adenine dinucleotide, oxidized form; LDH, lactic dehydrogenase; PK, pyruvate kinase; PEP, phosphoenolpyruvate; EGTA, ethylene glycol bis (@aminoethyl ether)N,N’-tetraacetic acid, ALV, asolectin vesicles; DTT, dithiothreitol. 0003.9861/92

53.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

PHOTOOXIDATION

OF SKELETAL

MUSCLE

Ca’+, adenine nucleotides, anthraquinones, and caffeine, and hence serves as a conformational probe for the Ca” release channel (6, 19). The present and accompanying study (22) demonstrates that photooxidation of the SR, using rose bengal as a sensitizer, results in rapid Ca2+ release from SR vesicles, leads to potent inhibition of high affinity [3H]ryanodine binding to its receptor, and alters the single channel characteristics of the Ca’-- release channel from skeletal muscle SR. The underlying molecular mechanism appears to involve an oxidation of histidyl residues on the SR Ca2+ release protein. Preliminary reports of these findings have been presented in abstract form (23). MATERIALS

AND

METH:ODS

Preparation of SR uesicles. For all flux studies, sarcoplasmic reticulum vesicles were prepared from rabbit hind leg and back muscle white skeletal muscle according to the method of MacLennan (24). The protein concentration was determined by absorption spectroscopy (25). For all [“Hlryanodine binding studies, SR vesicles from rabbit skeletal muscle were isolated by a minor modification of the method of Saito and coworkers (26) as described by Inui et al. (27). Protein concentration was determined in triplicate by the method of Lowry et al. (28) with bovine serum albumin as a standard, following removal of Hepes buffer by precipitating the protein with 2% perchloric acid, and dissolving the pellet in 1 M NaOH. Ca*+ efflux was measured from passively Measurement of Ca2’ eflux. loaded vesicles by dual wavelen;gth spectrophotometry using the Ca’+sensitive dye arsenazo III (ASIII; A675485“,) or antipyrylazo (APIII; A 72s790nm), or from actively loaded vesicles using a Ca’+-selective electrode (Cal-l- World Precision Instruments, Inc., New Haven, CT) (5). In passive experiments, SR vesicles (-10 mg/ml) were incubated in 100 mM KCl, 1-6 mM MgCl,, 1 mnn CaCl*, 50 mM Hepes-KOH, pH 7.0, overnight at 4°C. The vesicles were then diluted 50-fold into an identical Ca*+-free buffer containing 100 pM ASIII. Efflux was initiated by addition of the appropriate reagent. To determine the amount of releaseable Ca’+, 1 /*M A23187 was added upon completion of stimulated Ca*+ release. The Ca’+-AS111 signal was calibsrated with the addition of 4-8 PM Ca*+. For slower release experiments, SR vesicles were diluted into a buffer containing releasing reagent, then after a specified time 100 PM AS111 was added to establish an absorbance baseline. Subsequently, l-2 PM A23187 was added to release the remaining Ca*+. The percentage of Ca2+ remaining in the vesicles was determined by dividing the amount of Ca2+ remaining in the treated vesicles by the amount of releasable Ca2+ in the untreated vesicles. Active Ca*+ uptake was initiated by the addition of AcPO,. Ca2+ release was initiated by the addition of freshly prepared rose bengal or the histidyl reactive reagent ethoxyformic anhydride (EFA). Ca2+ efflux rates were determined from the maximal slope of the extravesicular Ca2+ concentration versus time. For those experiments carried out in an anaerobic environment, a buffer containing AcPO, and Ca*+ was deaerated by bubbling argon through the solution for approximately 20 min. Ca2+ uptake was initiated by addition of SR vesicles. The free Ca2+ concentration was monitored during Ca’+ uptake, and during the release phase following addition of rose bengal. In some experiments rose bengal was irradiated with a 360-W, broadspectrum light source (intensity of - 10,000 lux measured at the sample). A beaker of water was placed between the light source and the sample to screen out infrared light. In semidark experiments using rose bengal, the room was lit with diffuse light from an adjoining room. The rose bengal stock solution was stored in a dark bottle, and the reaction beaker was placed in a dark box. Hi&dine modifcation. Ethoxyformic anhydride (diethyl pyrocarbonate) reacts with the imidazole ring of histidine, yielding the products

SARCOPLASMIC

RETICULUM

513

N-ethoxyformylimidazole, ethanol, and carbon dioxide. N-Ethoxyformylimidazole has a characteristic spectrum with a maximum absorbance between 230 and 242 nm. The increase in the absorbance at 242 nm [e = 3.2 X lo3 M-’ cm-’ (30)] was used to quantitate the extent of histidine modification. SR vesicles (0.5-0.7 mg/ml) were suspended in 100 mM KCl, l-6 mM MgCIz, 50 mM Hepes-KOH, pH 7.0. Upon addition of EFA, the change in absorbance at 242 nm was followed as a function of time. The rate of formation of the chromophore was used to monitor the kinetics of histidine modification. ATPase activity was determined using a two-couATPase activity. pled-enzyme system (31), by measuring the initial rate of NADH oxidation (i.e., rate of decrease of A&. SR vesicles were irradiated in the presence of rose bengal for 1 min prior to dilution into the ATPase reaction buffer. Ca’+-dependent ATPase activity was reported as a percentage, and was calculated by dividing the ATPase of the rose bengal-treated sample by the ATPase activity of a sample containing no rose bengal. Asokctin uesicles. Asolectin vesicles were prepared by the following procedure. Asolectin, 60 mg, was diluted with 1.0 ml of distilled deionized H,O and homogenized into a uniform suspension. It was then purged with N, gas and sonicated for 15-20 min. After sonication, the asolectin vesicles suspension (ALV) was diluted 5-fold into a buffer containing 100 mM KCl, 50 mM Hepes, pH 7.0, buffer, 1 mM CaCl,. The vesicles were then frozen in liquid nitrogen, thawed at room temperature for 15 min, and sonicated for 30 s. Vesicles were then diluted 75-fold into a buffer containing 100 mM KCl, 50 mM Hepes, pH 7.0,3 mM MgCl, 100 pM AsIII. The internal Ca*+ content of the vesicle suspension was determined by adding A23187 (l-2 PM). Asolectin is a crude mixture of phosphatidylcholine-the primary lipid component of the sarcoplasmic reticulum. Calcium uptake of rose bengal-treated SR vesicles. Caz+ uptake was measured using the Ca’+-sensitive dye APIII. SR vesicles (0.2 mg/ml) were incubated in an uptake buffer (100 mM KCl, 1 mM MgClz, 50 mM Hepes, pH 7.0, 20 /IM CaCl,) in the presence or absence of rose bengal. The sample was then irradiated for 1 min. Following irradiation, 100 PM API11 was added. Active Ca2+ uptake was then monitored spectrophotometrically upon addition of 1 mM Mg’+-ATP. Upon completion of Ca*+ uptake, A23187 was added to release the accumulated Cast. The APIII/C!a2+ signal was calibrated by the addition of 2-4 PM Cal+. The percentage uptake activity remaining was determined by dividing the amount of Ca” uptake in the irradiated sample by the amount of Ca*+ uptake in the control sample (without rose bengal treatment). Singlet oxygen production was measured Singlet oxygen production. by the RN0 bleaching method described by Kraljic et al. (32). p-Nitrosodimethylaniline (RNO; 34 yM), imidazole (8 mM), and rose bengal (2 PM) were added to a KC1 (100 mM), Hepes (20 mM), pH 7.0, buffer. Samples were irradiated with a 360-W broad-spectrum lamp at a distance of 30 cm (shielded with a beaker of water). Aliquots were removed at various times and the A 44owas recorded. Controls in the dark and in the absence of sensitizer dye were performed. A decrease in absorbance occurs upon production of singlet oxygen. [31f]Ryanodine binding studies. Junctional membranes (20 pg/ml protein) were preincubated at 37°C for times ranging from 5 to 60 min in the presence or absence of 1 to 100 nM rose bengal (4 ml per concentration) in buffer containing 20 mM Hepes, pH 7.1,250 mM KCl, 15 mM NaCl, and 50 PM CaCl,. Uncapped glass scintillation vials containing each of the preincubation mixtures were irradiated using a broad-spectrum white light source having an intensity of 6500 lux measured at the top of the vial containing the assay mixture. In parallel experiments, preincubations with rose bengal were performed under minimal light. Following pretreatment with rose bengal, l-ml aliquots (in triplicate) of each reaction mixture were transferred to clean culture tubes under minimal light. [3H]Ryanodine was added to a final concentration of 1 nM, and incubated at 37°C for 2 h in complete darkness. The assays were quenched by rapid filtration as previously described (19). Each titration was repeated at least once.

514

STUART

ET AL.

In all figures and tables where no error bars are shown, the errors are less than 20% of the value displayed. Materials. All materials were purchased from Sigma Chemical Co. (St. Louis, MO) except for the following: dithiothreitol and Hepes buffer were purchased from Research Organics (Cincinnati, Ohio); imidazole was purchased from Aldrich Chemical Co. [sH]Ryanodine (60 Ci/mmolNew England Nuclear) was greater than 99% pure as assayed by HPLC. Commercial ryanodine (Penick Lot # 704RWP-1) was purified to greater than 99% purity by HPLC (33).

RESULTS

Sarcoplasmic reticulum vesicles, upon exposure to rose bengal, show an inhibition of Ca2+-dependent ATPase activity and active Ca2+ uptake (14-18). As shown in Fig. 1, Ca2+ uptake activity is more sensitive to rose bengal than is Ca2+-dependent ATPase activity. Ca2+ uptake shows a half maximal inhibition (I&,) at 0.1 PM for the light reaction (6.5 PM for the dark reaction) while ATPase activity has an IC50 of -0.5 I.IM for the light reaction (11 PM for the dark reaction). The enhanced sensitivity of active Ca2+ uptake as compared to Ca2+-dependent ATPase activity could be caused by either an uncoupling of active transport from ATPase activity or by an increase in the Ca2+ permeability of SR vesicles. In this report, the effects of photooxidation on the Ca2+ e&x properties of isolated skeletal SR vesicles are examined. At low concentrations (less than 1 PM), rose bengal causes rapid Ca2+ efflux from actively loaded SR vesicles when irradiated with a 360-W, broad-spectrum light source (N 10,000 lux) (Fig. 2C, trace 2). In the semidark, 1 PM rose bengal is significantly less effective in stimulating Ca2+ release (Fig. 2C, trace 3), while at higher concentrations rose bengal causes rapid release of Ca2+ without direct illumination. Experiments performed in the presence of ambient fluorescent light gave the same results as those performed in the semidark. The enhanced effectiveness of light in stimulating Ca2+ release from SR vesicles treated with rose bengal is further demonstrated in Figs. 2A and 2B, in which the rose bengal concentration dependence is shown. Ca2+ efIlux rates increase in a dose-dependent manner for both the light and the dark reactions. At concentrations of rose bengal greater than 1 PM, the difference between the light-dependent and the dark reaction is relatively insensitive to the concentration of rose bengal. At concentrations higher than 50 PM, rose bengal binds Ca2+ in a nonspecific fashion and forms a multimolecular aggregate (34). Therefore the use of high concentrations of rose bengal were avoided. Photosensitization of rose bengal in the presence of oxygen leads to the generation of short-lived reactive oxygen intermediates such as singlet oxygen and superoxide radicals (34). Rose bengal-stimulated singlet oxygen production was measured by the method of Kraljic et al. (32). In this method, the triplet state of rose bengal transfers its energy to molecular oxygen, resulting in singlet oxygen formation which then oxidizes imidazole to a transannular peroxide intermediate with the subsequent bleaching of

.ATPose

ochvily

0 Uoloke

[Rose

bengal],

pM

[Rose

bengal],

.M

FIG. 1. Ca2+ uptake and ATPase activity as a function of rose bengal concentration. SR vesicles were diluted into a buffer containing 100 mM KCI, 1 mM MgClz, 20 nM CaClz and 50 mM Hepes, pH 7.0 to a final protein concentration of 0.2 mg/mL. The indicated concentration of rose bengal was added and the vesicles were incubated for 1 minute in ambient light (Figure lb) or irradiated with a 360-watt, broad spectrum light source (Figure la). The treated SR vesicles were diluted into an ATPase (0) reaction buffer containing 150 mM KCl, 75 mM Hepes, pH 7.0, 1.5 mM MgC12, 10 mg/mL PEP, 10 mg/mL PK, 27 mg/mL LDH, 0.4 mg/mL NADH and 2 PM A23187 to a final protein concentration of 0.018 mg/mL. 1.8 mM ATP was added to start the reaction. The decrease of the absorbance at 340 nm was followed as a function of time. The percent ATPase activity remaining was calculated by normalizing the ATPase activity of the SR without the addition of rose bengal to be 100% activity. This represents a Ca2+ dependent ATPase activity of 3.35 + 0.10 ymoleslmg-min. Ca*+ uptake (0) was determined using APIII. 1 mM Mg’+-ATP was added to the treated vesicles to initiated calcium uptake. The percentage uptake was determined by dividing the amount of uptake with rose bengal treated vesicles by the amount of uptake with untreated vesicles (-100 nmoles Ca*+/mg SR).

p-nitrosodimethylaniline (RNO). In Fig. 3, we observe the production of singlet oxygen following irradiation of rose bengal under conditions that are the same as those used to induce rapid Ca2+ release from SR vesicles. In the absence of direct illumination no singlet oxygen is detected. If singlet oxygen production is responsible for rapid Ca2+ release from SR vesicles upon exposure to light, release observed in the dark apparently proceeds by a different mechanism. The involvement of oxygen in the light reaction was established by carrying out e&x experiments in both aerobic and anaerobic buffers. In the presence of light at 2 PM rose bengal, the Ca2+ release rate measured under anaerobic conditions was -20% of the rate measured in an aerobic environment. The release rate measured in the dark (at 10 PM) was unaffected by deaeration of the solution. In addition to triggering rapid Ca2+ release from actively loaded SR vesicles, rose bengal is shown to trigger Ca2+ release from SR vesicles passively loaded with Ca2’. Figure 4A shows that the amount of Ca2+ remaining in the vesicles decreases with increasing time of exposure to light and rose bengal. In Fig. 4B, SR vesicles were incubated with various concentrations of rose bengal for 5 min in the presence of light, and the amount of Ca2+ remaining in the vesicles was measured. The Ca2+ content of the vesicles is very sensitive to rose bengal at concentrations less that 50 nM. Between 50 and 100 nM rose bengal there

PHOTOOXIDATION

OF SKELETAL

MUSCLE

SARCOPLASMIC

515

RETICULUM

4

B 0 Light 0 Semi-dark

!

,

0

10

I

I

I

20

30

40

Rose Bengal

0 0

1

[uM]

90

180 TIME,

2 3 Rose Bengal [uM]

4

5

270

seconds

FIG. 2. Rose bengal-induced Ca2+ efflux from actively loaded SR vesicles. (A) SR vesicles were actively loaded with Ca*+ as described in Fig. 1. Following the addition of 20 PM Ca’+, uptake was initiated with 2.0 mM AcPO,. After accumulation of Ca’+, efflux was stimulated by the addition of various concentrations of rose bengal in the semi-dark (0) or in the presence of light (0). (B) shows the concentration dependence at low rose bengal concentrations. (C) Demonstrates the time dependence of active Ca*+ uptake and release induced by rose bengal. Efflux was initiated by the addition of either (1) 5 FM rose bengal in the semi-dark or (2) 1 PM rose bengal plus irradiation from a 360-watt light source at a distance of 30 cm. As a control, (3) 1 PM rose bengal was added in the semi-dark; (4) represents the baseline if no effluxing reagent is added. Extravesicular Ca2+ concentration is monitored using a Ca2+ selective electrode as described in Methods.

is no difference in the almount of Ca2+ released after 5 min of exposure to rose bengal (-70%). However, at 1 PM rose bengal all of the Ca2+ has been released within 5 min. This biphasic behavior appears to be due to an interaction with two types of binding sites. Modification of the highly reactive site caused release of 70% of the Ca’+, while the remaining 30% is released at higher concentrations of rose bengal. Unlike the earlier figures (Figs, 1, 2) in which Ca2+ had been actively accumulated, these experiments with passively loaded vesicles were carried out at significantly lower rose bengal concentrations and incubated with rose bengal for longer periods of time. The inclusion of high Mg2+ (5 mM) in the assay medium guar-

anteed that over the 5-min release phase, control untreated vesicles remained tight to Ca2+. The molecular mechanism underlying photoinduced oxidative damage to skeletal muscle SR remains to be clarified. Several amino acids are susceptible to oxidation, including cysteine, histidine, tryptophan, and tyrosine. It has previously been reported that histidine oxidation, via a singlet oxygen mechanism, proceeds more quickly than the photooxidation of other amino acids (35). Histidine, a known quencher of ‘02, is shown to inhibit rose bengalinduced Ca2+ efflux (Table I), in both the dark and the light. Moreover, a large excess of cysteine does not inhibit Ca2’ efIlux induced by rose bengal. These data are con-

516

STUART

ET AL.

. .

m

5 G

6 100 1. .

-.;

;

~-.4

-1-.

2

20 40

TIME. minutes

F’IG. 3. Singlet oxygen production by rose bengal. In a buffer containing 100 mM KCl, 20 mM Hepes, 8 mM imidazole, pH 7.0, 34 PM RNO, samples were exposed to rose bengal for the time period shown. The reaction was carried out in the dark (0 with 10 pM rose bengal); in the light (X with 2 PM rose bengal); and as a control in the absence of rose bengal in the light, 0. Following illumination and/or exposure to rose bengal, the absorbance at 440 nm was measured (30).

sistent with the involvement of histidyl residues in the photooxidation process. However, given the very large concentration of histidine required to partially inhibit rose bengal-induced Ca2+ release, further evidence is required to implicate a histidyl residue as a site of photooxidative damage. Rose bengal partitions into membranes and may induce its effect by increasing membrane fluidity. In order to determine the influence of rose bengal on the lipid components of the membrane, phospholipid vesicles made from asolectin (ALV) were loaded with Ca2+, rose bengal was added, and changes in the Ca2+ permeability of the vesicles were monitored. Using the Ca2+-selective dye antipyrylazo III the Ca2+ remaining in the vesicles after rose bengal treatment was measured. Rose bengal (10 PM in the dark) did not increase the permeability of the ALV to Ca’+. Incubation of the ALV with 3 PM rose bengal and irradiated for 1 or 2 minutes also resulted in no increase in the Ca2+ permeability of the ALV (data not shown). These results indicate that the action of the rose bengal is not likely to be caused by oxidation of the lipid component of the SR vesicles nor a nonspecific increase of lipid fluidity. The presence of adenine nucleotides stimulates sulfhydryl oxidation-induced (6, 10) and Ca2+-induced Ca2+ release (2,3,36). In an attempt to determine if the adenine nucleotide binding site might be the photodynamic target, the interaction between rose bengal-induced Ca2+ efllux and adenine nucleotide stimulation of Ca2+ release was monitored. At the high free Mg2+ concentrations used in these experiments (3 mM), in the absence of rose bengal, adenine nucleotides did not induce Ca2+ release. However, as seen in Table II, efflux rates induced by rose bengal in

100

1000

IROSE BENGALI. nM

FIG. 4. Rose bengal-induced Car+ efflux from passively loaded vesicles. SR vesicles were incubated on ice overnight in a medium containing 100 mM KCl, 5 mM MgClr, 1 mM CaCI,, 50 mM HEPES, pH 7.0. They were then diluted (50X) into an identical buffer containing no added Car+ with various concentrations of rose bengal. The samples were irradiated with a 360-watt light source at 30 cm for, the designated time. 100 PM As111 was then added, an absorbance baseline established (675685 nm). Subsequently, 1.0 PM A23187 was added to determine the amount of Ca*+ remaining in the vesicles. One aliquot of 4-8 PM Ca*+ was added to calibrate the AsIII-Ca*+ signal. The percentage Ca2+ remaining in the vesicles was calculated by normalizing to the amount of A23187 releasable Ca*+ from untreated SR vesicles as 100%. Figure 4A shows the amount of Ca2+ release as a function of time at a rose bengal concentration of (0) 10 nM, (0) 20 nM, (0) 50 nM and (W) 100 nM. Figure 4B shows the dependence on rose bengal concentration after incubation for 5 minutes.

the presence of 1.0 mM Mg’+--AMP-PCP were inhibited -40%. The presence of AMP-PCP appears to protect the photooxidation target. The absorbance spectrum of rose bengal in the visible range was unaltered by a large excess of AMP-PCP (not shown), indicating that protection from photooxidation was not likely to be caused by

TABLE Modification

Effluxing

I

of Rose Bengal-Induced by Amino Acids

reagent

17 PM rose bengal (dark) +40 mM histidine +40 mM cysteine 3 PM rose bengal (light) +40 mM histidine +40 mM cysteine +4OmM lysine

Efflux rate (nmol/mg-s)

11.3 f 8.0 k 13.1 f 4.2 + 2.1 + 4.6 f 4.1 f

1.1 0.4 3.0 0.3 0.6 0.2 0.01

Ca2+ Release

Percentage inhibition

-30% 0 -50% 0 0

Note. SR vesicles were actively loaded as described in the legend to Fig. 2. After accumulation of the added calcium, the reagant to be tested was added, then efflux was initiated by the addition of rose bengal. Percentage inhibition was calculated by comparing the Ca*+ efflux rate to the rate initiated by rose bengal alone. The standard deviation shown is for n > 3 data points.

PHOTOOXIDATION T.4BLE

OF SKELETAL

MUSCLE

II

Modulation by Mg2+ and AMP-PCP of Ca2+Release Induced by EFA and Rose Bengal Effluxing

reagent

17 pM rose bengal (dark) +1 mM Mg2+ AMP-PCP 3 pM rose bengal (light) +1 mM Mg*+ AMP-PCP 10 pM rose bengal (dark) +l mM Mg2+ +3 mM Mg2+ +6 mM Mg2+ EFA-induced WA]

(mM) 0.5 1.0 5.0

Efflux rate (nmol/mg-s)

% Inhibition

11.3 + 1.1 6.8 4.2 + 0.3 2.7 f 0.4

-40% -36%

3.8 A 1.6 3.6 k 0.8 4.3 f 0.8 Ca*+ release (3 mM Mg*+) Efflux rate (nmol/mg-min) 4.6 12.0 51.0

Mg2+ dependence of EFA (1.0 mM) induced release Pk*‘l

bM) 1.0 3.0 6.0

SARCOPLASMIC

517

RETICULUM

Light-dependent rose bengal inactivation of the ryanodine receptor was also found to be insensitive to the free Ca2+ concentration (If&,, at 1 and 50 I.LM Ca2+ both equal 5.8 nM), and to the presence of 1 mM DTT (If&, = 5.2 nM data not shown). It was shown in Table II that AMPPCP, a nonhydrolyzable analog of ATP, inhibits rose bengal-stimulated Ca2+ release from SR vesicles. Rose bengal also inhibited [3H]ryanodine binding in the absence of light, but significantly higher concentrations of the dye were required. A 30-min exposure of SR vesicles to 0.1, 1.0, and 6.0 WM rose bengal prior to the addition of 1 nM [3H]ryanodine resulted in a 2, 15, and 75% inhibition of high affinity ryanodine binding to its receptor, respectively. Pretreatment of SR with 1.0 mM AMP-PCP, a nonhydrolyzable analog of ATP, completely protected the SR from light insensitive inactivation by rose bengal. Nanomolar concentrations of rose bengal inhibit [3H]ryanodine binding to SR vesicles (Fig. 5A), and stimulate Ca2+ release from SR vesicles (Fig. 4B). Both of these assays were carried out following exposure to rose

Efflux rate (nmol/mg-min) 14.0 12.0 8.3

Note. SR vesicles were actively loaded as described in the legend to Fig. 2. After accumulation of the added calcium, the reagent to be tested was added, then efflux was initiated by the addition of either rose bengal or EFA. Percentage inhibition was calculated by comparing the Gas+ efflux rate to the corresponding rate initiated by rose bengal alone. The Ca2+ release rates initiated by :rose bengal are reported in nmol/mg-s, while those induced by EFA are expressed in nmol/mg-min. The standard deviation shown is for n > 3 data points.

% [%I Ryanodlne sound

;F .binding of rose bengal to the nucleotide. Moreover, unlike SH oxidation-induced (4) and Ca2+-induced Ca2+ efflux (3), which are strongly inhibited by millimolar concentrations of Mg’+, the rose bengal-induced Ca2+ e&x rate is unaffected by increasing external Mg2+ concentrations. Table II shows that at M:gzt concentrations as high as 6 mM, rose bengal-induced. Ca2+ efflux rates remain unaltered. Ruthenium red, another known inhibitor of Ca2+ release, was shown to inhibit rose bengal-induced stimulation of Ca2+ channel gating in bilayer reconstitution experiments (22). Ryanodine, at nanomolar concentrations, is known to specifically bind to the Cazf release protein from SR. In Fig. 5A, the effect of rose bengal on [3H]ryanodine binding to SR vesicles is examinjed. Exposure of SR membranes to 100 nM rose bengal in the absence of white light does not inhibit the binding of [3H]ryanodine in the subsequent assays, whereas 5-, 30-, and 60-min irradiations of SR in the presence of rose bengal give IC& values of 29, 8, and 4 nM (average of two determinations each in triplicate).

I

k

\

-II

\

k&-0-, I -8 Log Rose Bengal, M

1 -7

FIG. 5. [3H]ryanodine binding to the skeletal SR receptor is extremely sensitive to nanomolar rose bengal and correlates well with passive release from SR vesicles. (A) SR membranes (15-20 pg protein) were incubated at 37°C with the indicated concentrations of rose bengal in near complete darkness for 30 min (0) or exposed to white light (-6,500 lux) for 5 min (A), 30 min (0), or 60 min (A). Binding of 1 nM [3H]ryanodine was subsequently determined in complete darkness as described in Materials and Methods. Data shown are the mean values of triplicate determinations whose standard deviation (SD) was l pM which was diminished in the presence of 1 mM AMP-PCP. The similarity between the lightdependent and the dark reaction as far as modification of Ca2+ release and [3H]ryanodine binding suggests that the modified site on the release protein is the same for both reactions. However, given that the light reaction is O2 dependent and the dark reaction is O2 independent, the mechanism by which the site is altered is not the same for the two reactions. Tenu et al. (13) reported that the histidine modification of the SR (using EFA) that resulted in inhibition of Ca’+dependent ATPase activity was not at the ATP binding site on Ca2’, Mg2+-ATPase, but rather that histidine modification induces a conformational change resulting in altered ATP binding to the Ca2+ pump. In this paper, we observe that EFA-induced histidine modification results in release of Ca2’ from SR vesicles. Histidyl alter-

ET AL.

ation is also inhibited by the ATP analog AMP-PCP. In a similar fashion to the Ca2+ pump, adenine nucleotide binding may result in a conformational change that decreases the accessibility to this critical histidyl residue, associated with the Ca2+ release mechanism of the SR. The theory that the photooxidation site on the SR is a histidyl residue is supported by the observation that excess histidine in the efl-luxing buffer decreases Ca2+ efflux rates induced by rose bengal by -40%. Cysteine or lysine in the reaction buffer had no effect on the release rates. Also, when SR residues are photooxidized in the presence of rose bengal for 1 min, a strong absorbance maximum appears at -250 nm, which may be attributed to the photooxidation of histidine. At longer illuminations times tryptophanyl residues also appear to be modified. The photooxidation of 5 mM lysine or methionine does not produce an increased absorbance between 220 and 320 nm. The photooxidation of cysteine produced a decreased absorption at 240 nm, presumably from the production of cystine. The peak observed at -250 nm when the SR is photooxidized is likely caused, at least in part, by the photooxidation of histidyl residues. The peak at -306 nm which appears after significantly longer illumination time indicates that a slower photooxidation of tryptophan may also occur. Since photooxidation-induced Ca2+ efflux is essentially complete within 30 s, it is unlikely that the oxidation of the tryptophanyl residues is responsible for inducing rapid Ca2+ efflux. This is further supported by the observation that ethoxyformic anhydride, a known histidyl modifying reagent, also is effective in of inducing Ca2+ release from SR vesicles. Stimulation Ca2+ release by both EFA and rose bengal is inhibited by adenine nucleotides, and is relatively insensitive to millimolar concentrations of Mg2+. Although it is likely that the photooxidation target on the SR is a histidyl residue, other hypotheses should not be excluded. EFA is not absolutely specific for histidyl groups. Also, the spectrophotometric data presented (Fig. 6) describe rose bengal’s interaction with all SR protein components. The Ca2+ release protein is a relatively minor component which may be modified in a different manner, not identifiable by spectral changes. In conclusion, the results presented here support the hypothesis that photooxidation is acting on the Ca2+ release protein of the SR and that the histidyl residue(s) being modified are located close to the ATP binding site on the release protein. Further evidence supporting the interaction of rose bengal with the Ca2+ release protein is presented in the following paper (22).

ACKNOWLEDGMENT The authors thank Kristy nical assistance.

Gayman and Joseph Cronin for their tech-

PH’DTOOXIDATION

OF SKELETAL

SARCOPLASMIC

A. N. (1984) Physiol. Reu. 64, 1240-1320.

2. Nagasaki, K., and Kasai, M. (1983) J. Rio&em. (Tokyo) 94, llOl1109. 3. Meissner, G. (1984) J. Biol. Chm. 259, 2365-2374. 4. Trimm, J. L., Salama, G., and Abramson, J. J. (1986) J. Biol. C/rem.

261, 16,092-16,098. 5. Abramson, J. J., Cronin, J. R., and Salama, G. (1988) Arch. Biochem. Biophys. 263, 245-255. 6. Abramson, J. J., Buck, E., Salama, G., Casida, J. E., and Pessah, I. N. (1988) J. Biol. Chem. 263, 18,750-18,758. I. Nagura, S., Kawasaki, T., Taguchi, T., and Kasai, M. (1988) J. Biochem. 104,461-465. 8. Zaidi, N. F., Lagenaur, C. F., Abramson, J. J., Pessah, I., and Salama, G. (1989) J. Biol. Chem. 26,4, 21,725-21,736.

521

RETICULUM

21. Rousseau, E., Smith, J. S., and Meissner,

REFERENCES 1. Martonosi,

MUSCLE

G. (1987) Am. J. Physiol.

253,C364-C368. 22 Xiong, H., Buck, E., Stuart, J., Pessah, I. N., Salama, G., and Abramson, J. J. (1991) Arch. Biochem. Biophys. 292, 522-528.

23. 24. 25. 26.

Stuart, J., and Abramson, MacLennan, Kalckar,

J. J. (1989) Biophys. J. 56, 15a.

D. H. (1970) J. Biol. Chem. 245, 45084518.

H. M. (1947) J. Biol. Chem. 196,461-475.

Saito, A., Seiler, S., Chu, A., and Fleischer,

S. (1984) J. Cell Biol.

99,875-885. 27. Inui, M., Saito, A., Fleischer, S. (1987) J. Biol. Chem. 262, 17401747. 28. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 29. Chance, B., Legallais, Biochem. 66,498-514.

V., Sorge, J., and Graham, N. (1975) Anal.

R. J., Xiong, H., Abramson, J. J., and Salama, G. (1989) J. Biol. Chem. 264, 21,737-21,747. 10. Stuart, J., and Abramson, J. J. (1988) Arch. Biochem. Biophys. 264, 125-134.

30. Muhlrad, A., Hegyi, G., and Horanyi, M. (1969) Biochim. Biophys. Acta. 181,184-189. 31. Tipton, K. F. (1978) Techniques in Protein and Enzyme Biochem-

V. (1986) Biochem. J. 240, 509-517. 11. Shoshan-Barmatz, V. (1987) Biochem. J. 243, 165-173. 12. Shoshan-Barmatz, 13. Tenu, J. P., Ghelis, C., Leger, D. S., and Carreite, J. (1976) J. i?iol. Chem. 254, 4323-4329. 14. Yu, B. P., Masoro, R. J., and Bertrand, H. A. (1974) Biochemistry

32. Kraljic, J., Mohsni, S. E., and Arvis, M. (1978) Photo&em. Photobiol. 27,531-537. 33. Waterhouse, A. L., Holden, I., and Casida, J. E. (1984) J. Chem.

9. Zaidi, N. F., Lagenaur, C. F., Hilkert,

13,5083-5087. 15. Martonosi, A., Boland, R., and Halpin,

R. A. (1972) Cold Spring

Harbor Symp., Quant. Biol. 37, 455-468. 16. Kondo, M., and Kasai, M. (1974) Photo&em. Photobiol. 19,35-41. 17. Morris, S. J., Silbergeld, E. K., Brown, R. R., and Haynes, D. H. (1982) Biochem. Biophys. h’es. Commun. 104, 130661311. 18. Watson, B. D., and Haynee, D. H. (1982) Chem. Biol. Interact. 41,

313-325. 19. Pessah, I. N., Stambuk, R. A., and Casida, J. E. (1987) Mol. Pharmacol. 3 1, 232-238.

20. Meissner, G. (1986) J. Biol Chem. 260, 6300-6306.

istry, B112, pp. 5-9, Elsevier/North-Holland,

Amsterdam.

Sot. Chem. Commun., 1265-1266.

34. Neckers, D. C. (1987) J. Chem. Educ. 64,649-656. 35. Means, G. E., and Feeney, R. E. (1971) Chemical Modification Proteins,

Holden-Day,

of

San Francisco.

36. Morii, H., and Tonomura, Y. (1983) J. Membr. Biol. 66,1993-2001. 37. Valaitis, A. P., Roe, L. G., and Kemp, R. G. (1987) J. Biol. Chem. 262,5044-5048. 38. Tsai, C. S., Wand, A. J., Godin, J. R. P., and Buchanan, G. W. (1982) Arch. Biochem. Biophys. 217, 721-729. 39. Brocklehurst, K. (1979) Int. J. Biochem. 10,259-274. 40. Brocklehurst, K. (1974) Tetrahedron 30, 2397-2407. 41. Lee, P. C., and Rodgers, A. J. (1987) Photochem. Photobiol. 45, 7986.