Vol. 175, No. 3, 1991

BIOCHEMICAL

March 29, 1991

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 858-865

QUANTITATION OF RYANODINE RECEPTOR OF RABBIT SKELETAL MUSCLE, HEART AND BRAIN

Ernest0 Damiani, Gianantonio Tobaldin, Pompeo Volpe and Alfred0 Margreth

Centro di Studio per la Biologia e la Fisiopatologia muscolare, c/o lstituto di Patologia Universita’ di Padova, vla Trieste 75,35131 Padova, Italy Received

January

25,

generale,

1991

SUMMARY: The total number of high-affinity ryanodine receptor (RyR) binding sites present in skeletal and cardiac muscle and in brain tissue of the rabbit was determined by [3H]ryanodine binding to subfractions obtained by differential centrifugation of homogenates prepared in a low-ionic strength medium, containing 0.5% Chaps. In all three tissues at least 80% of sH]ryanodine binding was recovered in the total membrane (TM) fraction obtained ILy centrifuging between 650 g for 10 min and 120,900 x g for 90 min. Skeletal muscle displayed higher contents of highaffinity RyR sites (about 49 pmol/g wet wt) than heart and brain (about 12 pmol and 3.5 pmol/g wet wt, respectively). The affinity for ryanodine, as well as the affinity for Ca* + , in the absence or presence of Ca *+-releasing drugs (caffeine and doxorubicin) of TM from skeletal muscle, were found to be identical to those of purified terminal cisternae. As low as 1 g of tissue was sufficient to perform several experiments. B 1991

Academic

Press,

Inc.

Redistribution of Ca2+ between different cell compartments through mobilization from internal stores plays a key role in several functions of both muscle and nonmuscle cells (1). In both skeletal and cardiac muscle, the efflux of Ca2+ from the lumen of sarcoplasmic reticulum (SR) into the myofibrillar space occurs through a Ca2+-release channel, known to be selectively localized to the junctional membrane region of terminal cisternae (TC) (2). This Ca2+ -release channel is sensitive in vitro to Ca2+, caffeine and ryanodine (2), and it is therefore also known as ryanodine receptor (RyR). Recent lines of evidence indicate that the RyR is expressed in brain tissue (3, 4) and, perhaps, also in liver cells (5). Investigation of the RyR in purified membrane preparations obtained from whole tissue homogenates by differential and isopycnic sucrose density centrifugation (6) requires

the availability of large tissue samples, i.e. at least IO grams of tissue, in the

case of muscle. We describe here a simple procedure for isolating in membranebound form the RyR, corresponding to about 80-90% of the [3H]ryanodine binding 0006-291X/91 Copyright All rights

$1.50

0 1991 by Academic Press, Inc. of reproduction in any form reserved.

858

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activity present, from homogenates of skeletal and cardiac muscle, as well as of brain tissue, of the rabbit. Because of the low amount of tissue required (about 1 gram of fresh tissue), this procedure lends itself as an easy method for comparative analysis of the content of RyR both in different tissues, and in each tissue, in relation to physiological and pathological changes.

MATERIALS AND METHODS All chemicals were analytical grade and were obtained from Sigma Chemical Co (St. Louis, MO), Merck A.G. (Darmstadt, Germany), or Carlo Erba (Milan, Italy). 19, 21aH]ryanodine (30-60 Ci/mmol) was purchased from New England Nuclear. Adult, New Zealand albino rabbits were used. TC membranes were obtained from fast hindleg muscles, according to Saito e&$ (6), with the modifications of lnui et. (7). Total membranes (TM) were prepared from 1-2 g of either frozen or fresh adductor magnus (a pure fast-twitch muscie), and of fresh heart muscle (both ventricle and atria), and whole brain tissue (cerebrum, cerebellum and brain stem), as follows: tissue was minced with scissors and homogenized with 10 volumes (5 volumes in the case of brain) of 10 mM Hepes, pH 7.4, 20 mM KCI, 0.5% Chaps, 100 uM PMSF and 0.5 pg/ml leupeptin, using a conical glass homogenizer with a Teflon pestle. After centrifuging at 650 x g for 10 min, the pellet was re-extracted twice by homogenizing with the same medium, and centrifuging at the same speed. The low-speed sediment was resuspended in IO-15 ml of 10 mM Hepes, pH 7.4, 0.5 M NaCI. The supernatants were pooled and centrifuged at 120,000 x g for 90 min, to obtain the soluble fraction (SF) and the TM fraction. TM was resuspended in 1 ml of 0.3 M sucrose, IO mM imidazole, pH 7.4, 100 uM PMSF and 0.5 ugjml leupeptin. Protein concentration was determined according to Lowry&& (8) using bovine serum albumin as a standard. For [aH]tyanodine binding, 50-400 ug of protein were incubated in 200 ul of 10 mM Hepes, pH 7.4, 1 M KCI, 25 FM CaC12 for 30 min at 37’ C, at ryanodine concentrations ranging between 1 and 100 nM. Bound [aH]ryanodine was determined by Millipore filtration and liquid scintillation counting, as reported previously (9). SDS/polyacrylamide gel electrophoresis was carried out according to Laemmli (10). Slabs were stained with Coomassie blue, followed by Stains All. Electrophoretic transfer of proteins onto nitrocellulose and Western blotting were carried out as reported previously (11).

RESULTS AND DISCUSSION Figure 1 A shows the protein electrophoretic profile of the low-speed pellet, TM fraction and SF obtained by centrifugation of skeletal muscle homogenate. The predominant presence of myosin heavy chains and of actin appeared to be a characteristic feature of the low-speed pellet (lane l), consisting of purified myofibrils and in which SR proteins, e.g. calsequestrin (CS), were barely detectable. The TM fraction (lane 3), obtained by centrifuging between 650 x g and 120,000 x g, differed strikingly in protein composition from both the myofibrillar pellet (MP) (lane 1) and the SF (lane 2). The TM fraction, at the same time, shared several of the characteristic protein components of purified TC from the same type of muscle (lane 4), as shown 859

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AND BIOPHYSICAL

0

69 kDa

RESEARCH COMMUNICATIONS

B

I

2

3

4

kDa

I

2

3

4

R 200

-

110 97‘Pase

m-

43-

Fig.1. Panel A) Protein profile of subfractions obtained from rabbit fasttwitch skeletal muscle. Proteins were resolved by SDS-PAGE according to Laemmli (lo), using a 510% polyacrylamide linear gradient gel. Slabs were stained by Coomassie blue and then Stains All. Lanes: 1) MP; 2) SF; 3) TM; 4) TC. 100 yg of protein were loaded for each lane. Asterisks indicate blue-staining bands. MHC= myosin heavy chain; RyR= ryanodine receptor; CS= calsequestrin.

Panel 6) Immunological cross-reactivity with anti-(rabbit skeletal muscle as in panel A, and transferred RyR) antibody. Proteins were electrophoresed onto nitrocellulose. Blots were incubated with anti-RyR antibody (5 pg/ml) and then with anti-guinea pig IgG antibody, conjugated with alkaline phosphatase. 100 1(g of protein were loaded for each lane. Lanes: 1) MP; 2) SF; 3) TM; 4) TC from fast-twitch muscle.

by direct comparison, i.e. the 100 kDa Ca*+ -ATPase, the 64 kDa protein CS, and, most significantly, a high molecular weight component comigrating with the RyR. Identification of the same component with the RyR was substantiated by immunoblot data, using polyclonal antibody specific for the RyR of rabbit fast skeletal muscle (12) (Fig. 1 B). The RyR was observed only in the TM fraction (lane 3), but not in the MP (lane 1) or SF (lane 2), in overloaded (100 ug of protein) gels. The MP, TM fraction and SF were further compared for [3H]ryanodine

binding.

Table 1 shows that, in agreement with the electrophoretic and immunobiot data, [3H]ryanodine binding to TM fraction accounted for 90% of the summed activities in the three fractions, corresponding to about 49 pmoles of RyR sites/g wet wt. Fig. 2 shows the saturation isotherms of [3H]ryanodine binding for TC (panel A) and for TM (panel B) from rabbit fast-twitch

muscle. Scatchard

plot analysis of

[3H]ryanodine binding data shows that TC, as compared to the TM fraction, were about four fold enriched in RyR. On the other hand, the affinity for ryanodine (Kd) did 860

Vol. 175, No. 3, 1991

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1

Distribution of ryanodine binding sites in total membrane (TM) fraction, soluble sarcoplasmic fraction (SF) and myofibrillar pellet (MP) of fast-twitch skeletal muscle specific activity bmol/ma

total binding

or)

% of total

(omolja wet wtl

TM

3.9220.48

43.94L8.90

90

SF

0.03+0.01

1.21+0.53

3

MP

0.009+0.003

3.58k1.54

7

Determinationswere carried out on five different preparations.Values are given as mean + SEM. The yield of protein for TM was 10.1 f. 1.2 mg/g of wet wt. Valuesfor TM are B, x values, derived from Scatchard plot analysisof ryanodine binding curves, whrfk values for SF and MP were obtained at 100 nM [sH]ryanodine.

not differ significantly between the two membrane fractions (see also Table 2). Given the yield of TC protein (0.433 0.04 mg protein/g wet wt, n= lo), the amount of RyR recovered in a TC fraction represented only 14% of the total number of RyR sites (6.9 vs 49 pmol/g wet wt). It is well known that tyanodine binding to skeletal and cardiac muscle receptors is modulated by Ca2+ (13, 14). Table 2 shows that the half-maximal activation of

2.0 1.0. 0, 0

20

40

60

80

100

0

Ryanodine

20

40

60

80

100

(nM)

Fig. 2. Binding of [3H]ryanodine to TC (panel A) and TM (panel B) from fasttwitch muscle. Ryanodine binding was performed as described in Methods. Each data point representsthe average of duplicate determinations,and is given as specific binding, i.e. the difference between total binding and that measured in

the presence of 10 PM cold tyanodine. Bm x (pmol/mg protein) and Kd (nM) values were obtained from Scatchard plot andysrs (insets). 861

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TABLE 2 [3H]ryanocfine binding to TC and TM of fast-twitch skeletal muscle: BmBx,K,., and affinity for Can+ TC $Z/mg

protein)

TM

16.37+3.03

(4)

3.92kO.48

(5)

13.74k3.32

(4)

9.48+ 1.45 (5)

Stimulatory Ca2+ Fpca) no addition

6.35 (2)

6.26 (2)

+ 10 mM caffeine

7.33 (2)

7.19 (2)

+ 50 FM doxorubicin

6.95

7.04

BF

and Kd values for [sH]ryanodine binding were obtained by Scatchard plot anaysls of binding curves. Values are given as mean 2 SEM for the number of determinations reported in parenthesis. The Ca 2+-dependence of [sH]ryanodine binding was determined by experiments carried yt in the same medium, except that 1 mM EGTA, 0.03-I .I mM CaCls and 20 nM [ Hlryanodine were used. Free C$+ concentrations (O.Ol-1OOpM) were calculated by the computer program of Fabiato nd Fabiato (19), using an apparent Ca 2+ -EGTA association constant of 3.06~1 off M-r (20) which had been determined at 37’ C for a medium buffered at pH 7.0 with 10 mM Hepes, i.e under experimentaf conditions very similar to ours. represents the free Ca2+ concentration which gave a 50% stimulation of binding. Computer fit of the data was accomplished using commercially available software (ENZFIlTER, version 1.03, Elsevier-Biosoft).

[3H]ryanodine binding was attained at pCa values that were virtually the same for TC (6.35) and TM (6.26), and similar to those reported by Alderson and Feher (13) for ryanodine binding to cardiac SR. Table 2 further shows that caffeine (10 mM), as well as doxorubicin

(50 PM), i.e. at concentrations of either drug causing maximally

enhanced Ca2+ release from TC (Q), sensitized the ryanodine binding sites to activation by Ca2’ of TM and TC, to a similar extent, i.e. by more than one order of magnitude (14). We conclude from these results that the ryanodine binding properties of skeletal muscle receptor in TC isolated according to Saito et a/. (6), and in TM, prepared by our method, are essentially the same. Quantitation of specific, high-affinity RyR sites of other tissues has also been accomplished by this new procedure. The RyR is present in both heart and brain (13, 15, 4), possibly in lower amount (16, 4). Table 3 shows that it is possible to measure [3H]ryanodine binding to TM and final soluble supernatant of both heart and brain, and on the cardiac MP. Only the brain low-speed pellet was unsuitable for ryanodine binding measurements. It is noteworthy that, under identical experimental conditions, 862

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Vol. 175, No. 3, 1991

TABLE 3 Distribution

HEART

of ryanodine binding sites in rabbit heart and brain specific activity

total binding

ffmol/ma or)

romol/a wet wt)

TM

298332

9.31_t1.19

SF

7.836.1

0.96+0.75

MP

2822.0

2.00+0.28

% of total

76 8 16

BRAIN TM

83328

3.0630.84

89

SF

5326.2

0.3630.40

11

NP

n.m.

--

--

Determinations were carried out on three different preparations.Values are given as mean + ,jD. Values for TM are B,,, values, derived from Scatchard plot analysisof [ H]ryanodrne binding curves, all the other values were obtained at 100 nM [3H]ryanodine. Scatchard analysis of binding data on TM yielded G values of 1.93kO.22 nM and 3.iQ1.08 nM for heart and brain, respectively The yield of protein for TM was 31.3~2.7 and 37.927.6 mg of protein/g of wet wt for heart and brain, respectively. Abbreviations are as in Table 1, except that, in the case of brain, NP corresponds to the nuclear pellet and SF to soluble fraction. n.m. = not measurable. the apparent affinity for ryanodine binding was similar in heart and brain, and higher than that of skeletal muscle (see Tables 2 and 3). Northern blot analysis of rabbit brain and heart mRNA has indicated that the cardiac isoform is also expressed in brain (17). As in the case of skeletal muscle, cardiac TM displayed the highest specific activity and accounted for at least 80% of total RyR sites. Specific activity of cardiac TM was less than 10% that of skeletal muscle (Table 1). This is likely due to the higher degree of heterogeneity of the cardiac TM fraction, as indicated by the protein yield, which was three times higher than that from skeletal muscle (about 31 and 10 mg of protein/g wet wt, respectively). Mitochondria are the most likely source of non-SR membranes in cardiac muscle. Heart muscle contained about one fourth of RyR sites of skeletal muscle and three

times more than brain.

However,

given the

heterogeneous cell composition of the brain and the regional variations of the distribution of the RyR, (for example higher in the hippocampus than in the brain stem; see Ref. 4), our results do not rule out the possible existence of specific neurons highly enriched in RyR (18). 863

Vol.

175, No. 3, 1991

The availability

BIOCHEMICAL

of a simple

together with [3H]ryanodine several significant

for preparing

RESEARCH COMMUNICATIONS

TM from different cell types,

binding to the isolated membrane

properties

fraction indicating that

of the RyR are not affected by the same procedure,

might be able to overcome exclusively,

method

AND BIOPHYSICAL

the need of investigating

in highly purified

membrane

preparations

these properties, enriched

solely or

in Ryf?, such as

isolated TC, in the case both of skeletal and cardiac muscle. Noting

that the recovery

of RyR in purified TC is only 14% of the total RyR content per unit of fresh muscle tissue, on account of [3H]ryanodine

binding data, our procedure thus lends itself as a

complementary

approach,

together

with functional

actual number

of ryanodine-sensitive

studies, to the problem

Ca2+ -release channels

fibers, as well as in other types of cells. The information titration data on the TM fraction might be conceivably

in skeletal

of the muscle

that one can gather from RyR useful not only in comparative

studies between different tissues, as shown here, but also when investigating

age-

related or pathological

with

changes

in specific tissues, especially

when working

laboratory animals of small size.

Acknowledaments: This work was supported by grant funds from the Consiglio Nazionale delle Ricerche, and by grants (40% - 60%) from the Minister0 della Pubblica lstruzione to A.M.

REFERENCES 1. Meldolesi, J., Madeddu, L. and Pozzan, T. (1990) Biochim. Biophys. Acta 1055, 130-140. 2. Fleischer, S. and Inui, M. (1989) Annu. Rev. Biophys. Biophys. Chem. 18, 333-364. 3.Ashley, R.H. (1989) J. Membr. Biol. 111, 179-189. 4. McPherson, P.S. and Campbell, K.P. (1990) J. Biol. Chem. 265, 18454-18460. 5. Shoshan-Barmatz, V. (1990) FEBS Lett. 263,317-320. 6. Saito, A., Seiler, S., Chu, A. and Fleischer, S. (1984) J. Cell Biol. 99, 875-885. 7. Inui, M., Saito, A. and Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747. 8. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 9. Zorzato, F., Volpe, P., Damiani, E., Quaglino, D., Jr. and Margreth, A. (1989) Am. J. Physiol. 257, C504-C511. 10. Laemmli, U.K. (1970) Nature (London) 227,680-685. 11. Damiani, E., Spamer, C., Heilmann, C., Salvatori, S. and Margreth, Biol. Chem. 263, 340-343. 864

A. (1988) J.

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12. Zorzato, F., Chu, A. and Volpe, P. (1989) Biochem. J. 261, 863-870. 13. Alderson, B.H. and Feher, J. (1987) Biochim. Biophys. Acta 900, 221-229. 14. Ogawa, Y. and Harafuji, H. (1990) J. Biochem. 107, 894-898. 15. Inui, M., Saito, A. and Fleischer, S. (1987) 262, 15637-15642. 16. Inui, M., Wang, S., Saito, A. and Fleischer, S. (1988) J. Biol. Chem. 263, 1084310850. 17. Otsu, K., Willard, H., Khanna, V.K., Zorzato, F., Green, N.M. and MacLennan, D.H. (1990) J. Biol. Chem. 265, 13472-13483. 18. Ellisman, A.M., Deerick, T.J., Oujang, Y., Beck, C.F., Tanksley, S.J., Waiton, P.D., Airey, J. A. and Sutko, J.L. (1990) Neuron, 5, 135146. 19. Fabiato, A. and Fabiato, F. (1979) J. Physiol. (Paris) 75, 463-505. 20. Bers, D.M. (1982) Am. J. Physiol. 242, C404-C408.

Quantitation of ryanodine receptor of rabbit skeletal muscle, heart and brain.

The total number of high-affinity ryanodine receptor (RyR) binding sites present in skeletal and cardiac muscle and in brain tissue of the rabbit was ...
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