Transverse tubule calcium regulation in malignant hyperthermia

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 269, No. 2, March, pp. 497-506,1989

Transverse Tubule Calcium Regulation in Malignant Hyperthermia JAMES M. ERVASTI,* Depwtmsnts

of *Biochemistry

JAMES R. MICKELSON,t and ~Veler-inary

Biology, University

AND

CHARLES

F. LOUIS*~~’

of Minnesota, St. Paul, Minnesota 55108

Received August 12,1988, and in revised form November

‘7,1988

Transverse tubule (TT) calcium transport and permeability were examined in the inherited skeletal muscle disorder malignant hyperthermia (MH). ATP-dependent calcium uptake by TT vesicles isolated from normal and MH-susceptible (MHS) pig muscle had a similar dependence on ionized Ca2+ concentration (K,,, for Ca2+ of 0.21+ 0.04 and 0.25 t 0.05 /IM for MHS and normal TT, respectively), as well as a similar V,,,,, (20.9 -t 2.0 and 23.7 t 4.5 nmol Ca/mg protein/min for MHS and normal TT, respectively). Furthermore, the stimulation of calcium uptake by either calmodulin or CAMP-dependent protein kinase was similar in normal and MHS TT. Halothane concentrations greater than 2 mM inhibited calcium uptake by either normal or MHS TT to a similar extent (I&, = 8 mM). Dantrolene (10 PM), nitrendipine (1 PM), and Bay K 8644 (1 PM) had no significant effect on either the initial rates of calcium uptake or maximal calcium accumulation of either MHS or normal TT vesicles. However, in the absence of any added agents, maximum calcium accumulation by MHS TT was significantly less than by normal TT (90 + 10 versus 130 * 9 nmol Ca/mg protein after 15 min of uptake). This difference was not due to an increased permeability of MHS TT to calcium, nor was it due to a difference in the sarcoplasmic reticulum contamination (less than 5%) of the MHS and normal preparations. Although our results indicate there is no significant defect in MHS TT calcium regulation, the diminished maximum calcium accumulation by MHS TT may contribute to the abnormal sarcoplasmic calcium homeostasis in skeletal muscle during an MH crisis. 0 1989 Academic Press, Inc.

The primary defect in the skeletal muscle disorder malignant hyperthermia (MH)2 is believed to be an abnormality in the regulation of sarcoplasmic calcium (1). This hypothesis is supported by in vivo measurements of ionized Ca2+ concentrations, where the resting sarcoplasmic Ca2+ concentration has been reported to be elevated in MH-susceptible (MHS) human (2) and

pig (3) skeletal muscle; this resting Ca2+ concentration increases further during an MH episode (4). Recent studies of sarcoplasmic reticulum (SR) from MHS muscle have indicated a significant abnormality in the calcium release mechanism (5-11). However, the largest pool of calcium in skeletal muscle is in the extracellular space, where the millimolar calcium concentration requires the surface membrane to maintain a lO,OOO-fold calcium gradient. Thus, a defect in the surface membrane of MHS muscle, which renders it more permeable to calcium, or diminishes the efficiency of the calcium extrusion processes, could help to explain the reported elevation in sarcoplasmic calcium concentration. In support of this hypothesis, we have previously reported that sarcolemma (SL) isolated from MHS pig muscle has a significantly lower

’ To whom correspondence should be addressed at: Department of Veterinary Biology, University of Minnesota, 295 Animal Science/Veterinary Medicine Building, 1988 Fitch Avenue, St. Paul, MN 55108. ’ Abbreviations used: MH, malignant hyperthermia; MHS, malignant hyperthermia susceptible; TT, transverse tubule(s); SR, sarcoplasmic reticulum; SL, sarcolemma; EGTA, ethylene glycol bis (p-aminoethyl ether) N,N’-tetraacetic acid, NTA, N,N-bis(carboxymethyl)glycine; Pipes, piperazine-N,N’-bis[2ethanesulfonic acid]. 497

0003-9861/89 $3.00 Copyright All rights

0 1989 by Academic Press, Inc. of reproduction in any form reserved.

498

ERVASTI,

MICKELSON,

calcium transporting activity when compared to normal muscle SL (12). In contrast to the SL and SR, calcium regulation by the transverse tubule (TT) system has not yet been studied in MHS skeletal muscle. We have previously reported the isolation of TT vesicles from normal and MHS pig muscle and presented evidence indicating either an altered dihydropyridine receptor or decreased content of this protein in MHS TT (13). The TT dihydropyridine receptor has been implicated as the voltage sensor for the excitation-contraction coupling mechanism in skeletal muscle (14); however, there is also evidence to suggest that the dihydropyridine receptor functions as a calcium channel (15,16). Thus, a modified MHS TT dihydropyridine receptor could possibly result in an altered flux of extracellular calcium across the surface membrane and contribute to the abnormal MHS sarcoplasmic calcium concentration. To further investigate the role of the TT in MH, we have examined calcium transport by TT vesicles isolated from MHS and normal skeletal muscle. We found no difference between MHS and normal TT in the initial rate of calcium uptake, passive binding of calcium, passive calcium efflux, sodium-induced calcium efflux, or the effect of various agents on these processes. However, in agreement with our previous observations on isolated MHS muscle SL (12), the maximum amount of calcium that could be accumulated was significantly diminished in MHS TT vesicles. Our results indicate that minor abnormalities in MHS surface membrane calcium pumps may, in part, contribute to the abnormal regulation of sarcoplasmic calcium concentration in MHS muscle. EXPERIMENTAL

PROCEDURES

Materials. All pigs were obtained from the University of Minnesota Experimental Farm where they were tested for susceptibility to MH as previously described (10, 12). Halothane was from Halocarbon Laboratories, Inc. (Hackensack, NJ). Dantrolene was a gift of Norwich-Eaton Pharmaceuticals (Norwich, NY). Bay K 8644 and nitrendipine were gifts of Miles Laboratories, Inc. (New Haven, CT). Calmodulin was prepared from bovine testes as previously described

AND

LOUIS

(17). 45CaC1, and [3H]ryanodine were obtained from DuPont New England Nuclear. The ionophore A23187 was purchased from Calbiochem. Beef heart CAMPdependent protein kinase and all other reagents were obtained from Sigma Chemical Co. Water was redistilled from glass. Preparation of vesicles. TT vesicles were prepared by a modification of the method of Rosemblatt et al. (18). Briefly, crude muscle microsomes prepared aecording to Fernandez et aL (19) were loaded on tubes containing 25% (w/v) sucrose and centrifuged at 85,000g for 15 h in a swinging bucket rotor. The membrane fraction collected from the top of the 25% sucrose was used for all experiments. A detailed characterization of this fraction has been performed (13). SR vesicles were prepared by a modification of the procedure of Meissner (20) as described by Mickelson et al. (10). Determination of ionized Ca” concentrations. The ionized Ca2+ concentrations were calculated using the programs and constants described by Fabiato and Fabiato (21). p-Nitrophenylphosphatase activity. p-Nitrophenylphosphatase activity was determined at 37°C in 50 mM imidazole buffer (pH 7.5), 3 mM MgC12, 1 mM EGTA, and 5 mM p-nitrophenyl phosphate as described by Brandt et al (22). Calcium-dependent pnitrophenylphosphatase activity was defined as the difference between the activity measured in the absence and presence of 0.96 mM Call, (40 PM ionized Ca’+). [gHJRyandine binding assay. [3H]Ryanodine binding to TT preparations was measured as previously described for heavy SR (II), in a medium containing TT (0.2 mg protein/ml), 100 mM KCl, 10 mM Pipes buffer (pH 7.0), 10 nM [3H]ryanodine, 10 mM ATP, and a CaCiz-EGTA-NTA buffer set to give an ionized Ca2+ concentration of 6 pM (21). ATP-dependent calcium uptake. Calcium uptake by pig TT (0.03 to 0.10 mg/ml) was determined at 37°C in 120 mM KCl, 40 mM histidine buffer (pH 7.0), and 0.2 mM45CaC12 buffered with EGTA to produce defined ionized Cap+ concentrations, or 0.1 mM 45CaCl, alone (25 pM ionized Ca2+ (21)). Calcium uptake was initiated with 5 mM MgATP (sodium-free) and reactions were terminated by filtration through 0.45-hrn Millipore filters (type HAWP); the radioactivity of the filters was then determined. Bay K 8644 and nitrendipine were added to the uptake medium from lOO-fold stock solutions in absolute ethanol, while dantrolene was added from an aqueous stock solution (40 PM) which was made fresh daily. The effect of halothane on calcium transport was examined by adding halothane from a saturated aqueous solution (20 mM) through the Teflon septum of a glass Reacti-Vial (Pierce Chemical Co., Rockford, IL) 2 min prior to the initiation of calcium uptake. Passive calcium binding. Passive calcium binding to TT (0.03 mg/ml) was determined in 120 mM KCl, 40

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IIIM histidine buffer (pH 7.0), and 0.1 mM %aC& (100 GM ionized Ca2+ (21)). After incubation of TT for 15

min at 37”C, the membranes were filtered through 0.45-pm Millipore filters (type HAWP) and washed with 5 ml of ice-cold 120 mM KCl, 40 mM histidine buffer (pH 7.0); the radioactivity of the filters was then determined. Calcium efluz. Calcium efflux was measured by first actively loading TT vesicles with “%a in the presence of MgATP, as described above. After 15 min, efflux was initiated by the addition of 0.01 vol of 120 mM KCl, 40 mM histidine buffer (pH 7.0), 200 mM TrisEGTA (2 mM EGTA final concentration), and 1 mM Na orthovanadate (10 pM final concentration). The calcium retained by the vesicles was determined by filtration through Millipore filters. To measure the effect of sodium on calcium efflux, 45Ca-loaded vesicles were diluted with an equal volume of 40 mM histidine buffer (pH 7.0), 4 mM Tris-EGTA, and either 120 mM KC1 or 80 ITIM KC1 plus 40 mM NaCl (20 mM final sodium concentration). Sodium-dependent calcium efflux was defined as the difference between the amount of calcium retained by TT vesicles in the absence versus that retained in the presence of NaCl in the efflux medium. When the sodium concentration dependence of calcium efflux was studied, ionic strength was held constant by keeping the total [NaCl + KCI] = 120 mM. Statistical analysis. All comparisons of MHS and normal populations are expressed as means f SE and were analyzed by a Student’s t test.

RESULTS

Estimation

of SR ContamGnation of Pig TT

Vesicle preparations of known origin and purity were essential in order to accurately assess whether TT calcium transport was defective in MHS muscle. Of particular importance was the contribution which SR may make to the isolated TT preparations because of its high Cazf-ATPase and calcium pumping activities. Relative to isolated SR, TT preparations from a variety of species generally exhibit a high cholesterol content (23-25), low Ca’+-ATPase activity (23, 26), high (Na+ + Kf)-ATPase activity (23, 25, 26), and high ouabain binding (23, 27, 28) and dihydropyridine binding activities (27, 29). As previously reported (13), when compared to SR, the pig TT preparations used in this study have a 14-fold greater cholesterol content (1.08 pmol/mg protein), 1/15th the Ca’+-ATPase activity (69 nmol Pi/mg protein/min), high (Nat

CALCIUM

499

REGULATION TABLE

I

CALCIUM-DEPENDENTP-NITROPHENYLPHOSPHATASE ACTIVITY~OFPIGSKELETALMUSCLE TT AND SR VESICLES

Basal + Calcium Calcium dependent % SR contaminationb

Normal

MHS

(nT5j

(nT5)

25.4 k 0.8 26.9 k 1.0

28.0 f 1.6 28.6 f 1.5

13.1 f 0.6 45.2 f 2.3

1.5 -+ 0.9

0.6 + 0.7

32.1 t 2.2

4.7 f 2.8

1.9 + 2.2

(ZS)

“Means +- SE expressed in nmol p-nitrophenol formed/mg proteinlmin at 37°C. *Estimation of SR contamination was made with pthe assumption that all of the calcium-dependent nitrophenylphosphatase activity measured in TT was due to SR contamination.

+ KC)-ATPase activity (31 pmol/mg protein/h), high [3H]ouabain binding (100 pmol/mg protein), and 13-fold greater [3H]nitrendipine binding (26 to 40 pmol/mg protein). None of these parameters, however, gives an indication of the extent of SR contamination in the preparations. It has been reported that the SR Ca2+-ATPase can utilize p-nitrophenylphosphate as substrate, in contrast to the surface membrane Ca2+-ATPase (22), so this activity can be used to assess SR contamination in surface membrane preparations. Assuming all of the activity present in TT vesicles is due to SR contamination, the p-nitrophenylphosphatase activity of pig TT (Table I) indicated that SR contamination of TT was less than 5%. Calcium precipitating anions such as phosphate and oxalate have been reported to greatly stimulate SR calcium uptake by lowering the intravesicular calcium concentration and thereby lessening the calcium gradient (30). On the other hand, rabbit TT vesicles are reported to be much less permeable to oxalate and phosphate, and thus TT calcium uptake is not stimulated by these anions (22,31). Calcium uptake by SR was stimulated over loo-fold by 30 mM phosphate, whereas TT calcium uptake

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ATP-Dependent

2500

-rl-.=,c.;. 0

10

20

30

40

50

[Phosphate] (mM) FIG. 1. Effect of phosphate concentration on pig TT and SR calcium uptake. Calcium uptake by TT and SR vesicles was measured as described under Experimental Procedures in the presence of 0.1 mM 45CaC12 (25 pM Cat+) and the indicated concentration of phosphate. Two minutes after the addition of MgATP, calcium uptake was terminated and the amount of calcium retained by the vesicles was determined. Points represent the means of duplicate determinations.

was little affected by phosphate concentrations as high as 50 mM (Fig. l), indicating that SR contamination of porcine TT was negligible. In agreement with these results, 1 to 5 mM oxalate had little effect on TT calcium uptake while SR calcium uptake was greatly stimulated (data not shown). The final estimate of SR contamination of the pig TT preparations was provided by the measurement of [3H]ryanodine binding activity; ryanodine has recently been identified as a specific ligand for the SR calcium release channel (32-35). rH]Ryanodine binding in three normal and three MHS TT preparations was 0.32 + 0.10 and 0.22 * 0.10 pmol/mg protein, respectively. [3H]Ryanodine binding to pig heavy SR is typically 9-10 pmol/mg protein,3 which is in very close agreement with the binding to rabbit heavy SR under identical conditions (36), indicating that the SR contamination of pig TT was 3% or less. Thus the methods used to determine SR contamination of pig TT indicated that the TT preparations comprised no more than 5% SR.

3 J. R. Mickelson,

unpublished

observations.

Calcium Uptake by Pig TT

To examine whether contamination of our TT preparations by SR would interfere with the measurement of initial rates of TT calcium transport in the absence of calcium precipitating anions, calcium uptake was measured in TT preparations that included varying amounts of added SR (Fig. 2). The rate of calcium uptake by such preparations was linear between 0.5 and 2.0 min, even when SR comprised 20% of the total membrane protein. In the absence of any calcium precipitating anions, calcium accumulation by purified SR is maximal by 0.5 min, reaching values of 50 to 120 nmol Ca/mg protein (37-39) with little additional calcium uptake for up to 15 min (37,39). These results indicate that even in the presence of small amounts of SR contamination, the initial rates of TT vesicle calcium uptake could be accurately determined between 0.5 and 2.0 min. The initial rate of calcium transport by normal and MHS TT had a similar dependence on ionized Cazt concentration (K,,, for Ca’+, 0.25 + 0.05 and 0.21 + 0.04 PM for normal and MHS TT, respectively) as well as a similar V,,, (23.7 -+ 4.5 and 20.9 f 2.0 nmol Ca/mg protein/min for normal and MHS TT, respectively; Fig. 3). These values

60 50 2 t3

40

2 5

30

d % 3

20

d

10

1 .o

0.5 Time

1.5

2.0

(minutes)

FIG. 2. Effect of exogenously added SR on the rate of calcium uptake by pig TT. Calcium uptake was measured as described under Experimental Procedures in the presence of 0.1 mM %aC12 (25 pM Ca*+) and varying amounts of SR. The amount of SR added is expressed as a percentage of the total membrane protein (TT + SR) in the tube. Points represent the mean of duplicate determinations.

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CALCIUM

30 2 E

25

P z 0

20

E c

15

n+l

10

3 E

5

0 n

0 , 10-z

10’

10”

10-l

Normal MHS

1

Pa*+1 (PM) FIG. 3. Effect of ionized Ca2+ on the initial rate of calcium uptake by pig TT. Calcium uptake was measured as described under Experimental Procedures. The Ca*+ concentrations were maintained by a CaCl,EGTA buffer system. Each point is the mean + SE for three different MHS and three different normal TT preparations. The calculated V,,, values were 20.9 f 2.0 and 23.7 f 4.5 nmol Ca/mg proteinlmin, while the K,,* for Caa+ values were 0.21 f 0.04 and 0.25 * 0.05 pM for MHS and normal TT, respectively.

are in close agreement with those reported for rabbit TT and porcine SL vesicles (12, 31). However, when the time course of TT calcium uptake was studied in a medium containing 0.1 mM CaCl, (25 FM ionized Ca2+ (21); Fig. 4A), for a large number of

150 (

1

-m +-

5

501

REGULATION

preparations, maximum accumulation of calcium by MHS TT was only 69% of normal TT (90 * 10 versus 130 * 9 nmol calcium/mg protein respectively after 15 min, P < 0.005). Futhermore, over a wide range of ionized Ca2+ concentrations (0.053-100 PM ionized Ca2+), maximum calcium accumulation by MHS TT was only 60% that of normal TT. There was no difference in passive calcium binding between MHS and normal TT (3.05 + 0.32 and 3.56 & 0.45 nmol Ca/mg protein, respectively, n = 3). The effects of a number of agents which have been proposed to affect surface membrane calcium pump activity or calcium permeability were also studied (Table II). These agents were tested in the presence of the half-maximally stimulating ionized Ca2+ concentration (0.22 PM) in order to detect potential effects on K,,, or V,,, values for calcium transport activity. MHS and normal TT activities were stimulated to a similar extent by either exogenous calmodulin (1 PM) or CAMP-dependent protein kinase (0.1 mg/ml) and CAMP (5 PM). Dantrolene, the only clinically available drug for treatment of MH, had no effect on calcium uptake by either normal or MHS TT (Table II). Similarly, the calcium channel antagonist nitrendipine (1 PM) and agonist Bay K 8644 (1 PM) had no significant

200

e

Normal MHS

10

Time (minutes) FIG. 4. Time course of calcium

[Ca*‘l (PM)

uptake (A) and effect of ionized Ca2+ on maximum calcium accumulation (B) by pig TT. (A) Calcium uptake was measured as described under Experimental Procedures in the presence of 0.1 mM 4SCaClZ (25 pM ionized Cazf). Each point is the mean f SE for 17 different normal and 12 different MHS TT preparations. (B) The ionized Ca*+concentrations were maintained by a CaCI,-EGTA buffer system and TT calcium uptake was measured at 15 min as described under Experimental Procedures. Each point is the mean f SE for four different MHS and four different normal TT preparations.

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effect on calcium uptake (Table II). Furthermore, when calcium uptake by both normal and MHS TT was examined in the presence of halothane, calcium transport was inhibited in a similar fashion by halothane concentrations greater than 2 mM (I(& = 8 mM; Fig. 5). Calcium

Eflux

from Pig TT

The decreased calcium uptake of MHS TT described in Fig. 4 could be the result of a greater calcium permeability of MHS TT relative to normal TT. To examine this possibility, TT were first loaded with calcium for 15 min when calcium efflux was initiated by the addition of 0.01 vol of 120 mM KCl, 40 mM histidine buffer (pH 7.0), 200 mM EGTA, and 1 mM sodium orthovanadate (Fig. 6A). If the calcium ionophore A23187 was also added, essentially all of the loaded calcium was released (Fig. 6A), indicating that the calcium was sequestered in a vesicular pool. The slow rate of calcium release from calcium-loaded TT vesicles upon addition of EGTA and vanadate (Fig. 6A) was similar to that previously reported for MHS and normal SL (12), whereas the rate of calcium efflux from isolated SR is significantly greater (36). When normal and MHS TT were compared (Fig. 6B), no difference in calcium efflux rates were apparent. Linear correlation analysis of the log percentage calcium retained versus time, for each TT preparation, demonstrated that the slow phase of calcium efflux was approximately linear from 2 to 15 min after the addition of EGTA plus orthovanadate (r,, = -0.963). Thus, first-order rate constants of 0.0174 f 0.0014 and 0.0173 + 0.0024 min-’ were obtained for the slow phase of MHS and normal TT calcium efflux, respectively. Furthermore, there was no difference in the calcium efflux rates of MHS and normal TT when measured in the presence of 1 pM Bay K 8644 or nitrendipine (data not shown). Sodium-Dependent

Calcium Eflux

To examine whether TT sodium-dependent calcium efflux was abnormal in MHS muscle, TT vesicles were first loaded with 45Ca in the presence of MgATP. The cal-

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cium-loaded TT were then diluted with an equal volume of either 120 mM KCl, or 80 mM KC1 plus 40 mM NaCl (20 mM NaCl final), both containing 5 mM MgATP, 40 mM histidine buffer (pH 7.0), and 4 mM EGTA. As illustrated in Fig. 7A, more calcium was released from pig TT in the presence than in the absence of NaCl in the efflux medium; this enhanced calcium efflux in the presence of sodium was found to be linear for 7 min (not shown) after which the rate of efflux mirrored that in the absence of sodium. Both the rate (Fig. 7B) and the total amount of this calcium efflux was dependent on the final sodium concentration of the efflux medium. Normal and MHS TT were similar in the maximal rate of sodium-dependent calcium efflux (3.1 + 0.5 and 2.9 + 0.3 nmol Ca/mg protein/min for normal and MHS TT, respectively) as well as the total amount of calcium released (22 + 3.5 and 21 f 2.1 nmol Ca/mg protein, respectively, after 7 min of efflux). DISCUSSION

In this report, we present evidence that TT vesicles isolated from MHS pigs accumulate significantly less calcium than do TT from normal pigs (Fig. 4A). However, this difference between MHS and normal TT does not appear to be due to altered kinetics of the TT calcium pump because normal and MHS TT have a similar V,,, (23.7 + 4.5 and 20.9 + 2.0 nmol Ca/mg protein/ min for normal and MHS TT, respectively) and calcium concentration dependence for ATP-dependent calcium uptake (Kijz for Ca2+ of 0.25 rf- 0.05 and 0.21 + 0.04 pM for normal and MHS TT, respectively; Fig. 3). Futhermore, calmodulin and CAMP-dependent protein kinase, which independently regulate surface membrane calcium pumps, stimulated the rate of calcium uptake by normal and MHS TT in a similar fashion (Table II). The calcium permeability of MHS TT, as determined by passive calcium efflux from calcium-loaded TT, also did not appear to differ from that of normal TT (Fig. 6B). Although the difference between MHS and normal TT maximal calcium accumulation was measured over a wide range of ionized Ca2+ concentrations (0.053-100 pM

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CALCIUM

TABLE

503

REGULATION

II

THEEFFECTOFVARIOUSAGENTSONCALCIUMUPTAKEBY PIGTT Initial

Control + 0.1 mg/ml CAMP-PK 5 PM CAMP +l@ICaM

MHS

Normal

Agent tested

9.4 f 0.3 11.1 * 0.9

and

+ 10 I.~MDantrolene

Uptake at 15 min*

rate&

8.1 rt 1.5 10.1 + 2.0

UW

(125)

13.7 222.2 (148) 10.1 f 0.6

14.2 + 3.1 (173) 9.5 f 2.0

(116)

UW

+ 1 WMNitrendipine

9.0 f 0.7 (97)

+l fiMBayK8644

lO.l+ 0.8

7.5 -c 1.4 (95) 8.3 k 1.5

(107)

(102)

Normal 83.3 + 2.5 91.2 + 5.9 (110) 110 + 7.8 (131) 84.0 f 6.3 (101) 84.3 f 2.2 (101) 87.0 + 4.2 (104)

MHS 66.5 f 14.3 76.4 f 15.5

(116) 85.9 f 17.3 (130) 65.1 + 13.5 (99) 66.7 f 13.5 (101) 60.9 + 11.5 (92)

Note. Expressed as the means f SE in “nmol Ca/mg protein/min or *nmol Ca/mg protein for four normal and three MHS TT preparations. Numbers in parentheses are the values expressed as a percentage of the control value (100%). The Ca2+ concentration was maintained at 0.22 HIMwith a CaCla-EGTA buffer.

ionized Ca2+), it should be noted that this difference was not statistically significant in Fig. 4B. The most likely explanation could be the small sample number tested (n = 4 in Fig. 4B versus n = 12-17 in Fig.

40 10-l

0

10'

[tlalott~~nOe] (mM)

FIG. 5. Effect of halothane on calcium uptake by pig TT. Calcium uptake in the presence of varying concentrations of halothane was measured as described under Experimental Procedures. TT were incubated with the indicated concentrations of halothane for 2 min prior to the addition of MgATP. Samples were filtered at 1.5 min. The final Ca2+ concentration was 0.22 pM. Each point is the mean f SE of three different normal and three different MHS preparations expressed relative to each preparation’s control value in the absence of halothane (100%).

4A), as well as the preparation-to-preparation variability and relatively small (3040%) difference in calcium accumulation between MHS and normal TT. It is possible that normal TT accumulate significantly more calcium than does MHS TT as a result of a greater contamination of normal TT preparations by SR. SR calcium pump activity is much greater than that of TT so even a small contamination of TT by SR could obscure measurement of TT calcium transport rates. However, estimates of SR contamination reported here (Table I, Fig. 1, and the [3H]ryanodine binding data) suggest that SR contamination was no greater than 5% in either normal or MHS TT. An alternative explanation for the difference between MHS and normal TT maximal calcium accumulation could be that it is an artifact of the preparative method used. Muscle from MHS pigs often undergoes rapid postmortem changes (40) which can lead to damage of isolated membrane fractions (41). However, the MHS and normal TT vesicles used in this study had very similar cholesterol and phospholipid contents, [3H]ouabain and rH]saxitoxin binding, (Na+ + K+)-ATPase, Ca2+ATPase, and ouabain-sensitive, K+-stimulated p-nitrophenylphosphatase activities (13) demonstrating that the differences in

504

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120 100 = E

100

' E E r

80

-1 E 6 p $

60

2

2

90

MHS Normal

80

.$

40

+ -A-

S d

a-E-

z

c EGTA + A23187

z M

20

70

d

0

60 10

15

20

25

30

Time (minutes)

0

5

10

15

Time (minutes)

FIG. 6. Calcium efflux from pig TT. TT were actively loaded with %a and calcium efflux was induced at 15 min as described under Experimental Procedures. The points in a representative experiment (A) are the mean values of triplicate determinations. At 15 min either 2 mM EGTA and 10 pM sodium orthovanadate (+EGTA) or 5 FM A23187 (fA23187) was added. Comparison of calcium efflux in normal and MHS TT is shown in (B). At the indicated times after addition of EGTA plus orthovanadate, the calcium remaining in the TT was determined and expressed relative to the initial calcium load after 15 min of calcium uptake (100%). Points represent the means ? SE of seven different normal and seven different MHS TT preparations.

calcium accumulation were not preparation-generated artifacts. Since isolated TT vesicles must be in a sealed inside-out orientation for the calcium pump to sequester calcium, the difference between MHS and normal TT in maximal calcium accumulation could also be due to a decreased proportion of sealed inside-out vesicles in the MHS TT preparations. However, latency studies of (Na+ + K’)-ATPase activities, [3H]ouabain binding, [3H]saxitoxin binding, and p-nitrophenylphosphatase activities of these preparations indicate that both normal and MHS TT are composed of approximately 70% sealed inside-out vesicles, while 30% are leaky (13). A recent study of surface membranes isolated from MHS pig muscle by Rock and Kozak-Reiss (42) reported that in vitro, Ca’+-ATPase activity of MHS surface membranes was more sensitive to halothane which also rendered MHS surface membranes significantly more permeable to calcium than normal surface membranes. However, the (Na+ + K+)-ATPase activity of these MHS surface membrane preparations (42) was only one-fourth that reported for the normal surface membranes and the calcium uptake reached a value of only 15-19 nmol Ca/mg protein. Furthermore, the SR contamination of this

preparation was not determined, and its high Ca2’-ATPase activity is more characteristic of previously described SR preparations (10). Halothane has been observed to increase the extent of calcium release in both normal and MHS SR vesicles (10). Thus the results of Rock and Kozak-Reiss suggesting a major defect in MHS surface membranes (42) could be explained by a surface membrane preparation that was significantly contaminated with SR, which would be expected to release more calcium in the presence of halothane than would a highly purified surface membrane preparation. In addition to confirming our previous observations on MHS SL, the present study of calcium transport by MHS TT also addressed the possibility of a defect in the sodium-calcium exchange mechanism of MHS TT. That the sodium concentration has been previously reported to be elevated in MHS pig muscle led to the proposal that sodium-calcium exchange is altered in this syndrome (43). As previously reported for isolated cardiac (44) and skeletal (45) muscle surface membranes, we observed a slow sodium-induced calcium efflux in actively loaded pig TT (Fig. 7A). This sodium-induced calcium efflux has been attributed to the presence of a sodium-calcium ex-

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CALCIUM

505

REGULATION

160 2

140

5 5

120

E

g d s s

100

d

60

80

-c+ *

Control + 20 mM NaCl NaCl I

40 10

15

.

I

I

20

25

.

I 30

0

Time (minutes)

5

10

15 [NaCl]

20

25

30

(mM)

FIG. 7. Sodium-induced calcium efflux from pig TT. At time 0, TT calcium uptake was initiated with 5 mM MgATP. After 15 min of calcium uptake, calcium efflux was induced as described under Experimental Procedures. The points in a representative experiment (A) are the mean values of triplicate determinations. In addition to 2 mM EGTA and 10 PM sodium orthovanadate, the efflux medium contained a final concentration of either 120 mM KC1 (-NaCI) or 100 mM KC1 plus 20 mM NaCl (+20 mM NaCl). The sodium concentration dependence of the rate of this calcium efflux is shown in (B).

changer in these preparations (44, 45). However, MHS and normal TT did not differ with respect to the rate of this sodium-dependent calcium efflux, suggesting that sodium-calcium exchange is not abnormal in MHS muscle. A recent study of lymphocytes from MHS humans and pigs reported that very high (PM) concentrations of the calcium channel antagonist nifedipine inhibited a halothane-dependent increase in lymphocyte intracellular calcium concentration (46). This observation led to the proposal that dihydropyridine-sensitive calcium channels may be abnormal in MH (46). Regarding this possibility we have recently reported abnormal [3H]nitrendipine binding in MHS TT and MHS muscle homogenates (13). However, the present results do not support the proposal that a defect in the dihydropyridine-sensitive calcium channel renders the MHS TT membrane more permeable to calcium, as relative to normal TT neither Bay K 8644 nor nitrendipine had a significant effect on MHS TT calcium uptake or calcium efflux. In conclusion, we have presented evidence indicating that maximum calcium accumulation by MHS TT was less than that of normal TT. However, as with the defect in calcium transport in MHS SL (12), it appears unlikely that this MHS TT

defect contributes significantly to the elevated intracellular calcium concentration that has been reported in human (2) and pig (3) muscle, because the most significant difference between MHS and normal calcium uptake is observed only at very high calcium concentrations. On the other hand, it is possible that the deficiencies in MHS TT and SL calcium transport may contribute to the severity of an MH crisis. During such an episode, intracellular Ca2+ rises to high levels (4), so the MHS surface membrane calcium pumps may not be able to extrude calcium rapidly enough to offset the abnormal SR calcium release (5-11) of MHS muscle. ACKNOWLEDGMENTS The authors thank Dr. William Rempel for supplying the pigs used in this study and Mr. Michael Claessens for excellent technical assistance. This work was supported by National Institutes of Health Grant GM-31382. REFERENCES 1. GRONERT, G. A. (1986) in Myology (Engel, A. G., and Banker, B. Q., Eds.), pp. 1763-1784, McGraw-Hill, Minneapolis, MN. 2. LOPEZ, J. R., ALAMO, L., CAPUTO, C., WIKINSKI, J., AND LEDEZMA, D. (1985) Muscle Nerve 8,355358.

506

ERVASTI,

MICKELSON,

3. LOPEZ, J. R., ALAMO, L. A., JONES, D. E., PAPP, L., ALLEN, P. D., GERGELY, J., AND SRETER, F. A. (1986) Muscle Nerve 9,85-86. 4. LOPEZ, J. R., ALLEN, P. D., ALAMO, L., JONES, D., AND SRETER, F. A. (1988) Muscle Nerve 11,8288. 5. ENDO, M., YAGI, S., ISHIZUKA, T., HORIUTI, K., KOGA, Y., AND AMAHA, K. (1983) Biomed. Res. 4,83-92. 6. TAKAGI, A., SUNOHARA, N., ISHIHARA, T., NoNAKA, I., AND SUGITA, H. (1983) Muscle Nerve 6,510-514. 7. NELSON, T. E. (1983) J. Clin. Invest. 72,862-870. 8. OHNISHI, S. T. (1987) Biochim. Biophys. Acta 897, 261-268. 9. KIM, D. H., SRETER, F. A., OHNISHI, S. T., RYAN, J. F., ROBERTS, J., ALLEN, P. D., MESZAROS, L. G., ANTONIU, B., AND IKEMOTO, N. (1984) Bie chim. Biophys. Acta 7X,320-327. 10. MICKELSON, J. R., Ross, J. A., REED, B. K., AND LOUIS, C. F. (1986) Biochim. Biophys. Acta 862, 318-328. 11. MICKELSON, J. M., GALLANT, E. M., LITTERER, L. A., JOHNSON, K. M., REMPEL, W. E., AND LOUIS, C. F. (1988) J. Biol. Chem. 263, 93109315. 12. MICKELSON, J. R., Ross, J. A., HYSLOP, R. J., GALLANT, E. M., AND LOUIS, C. F. (1987) B&him Biophys. Acta 897,364-376. 13. ERVASTI, J. M., CLAESSENS, M. T., MICKELSON, J. R., AND LOUIS, C. F. (1989) J. Biol. Chem., in press. 14. RIOS, E., AND BRUM, B. (1987) Nature (London) 325,717-720. 15. CURTIS, B. M., AND CATTERALL, W. A. (1986) Bip chemistry 25,3077-3083. 16. FLOCKERZI, V., OEKEN, H.-J., HOFMANN, F., PELZER, D., CAVALIE, A., AND TRAUTWEIN, W. (1986) Nature (London) 323,66-68. 17. GOPALAKRISHNA, R., AND ANDERSON, W. B. (1982) B&hem. Biophys. Res. Commun. 104,830-836. 18. ROSEMBLATT, M., HIDALGO, C., VERGARA, C., AND IKEMOTO, N. (1981) J. Biol. Chem. 256, 81408148. 19. FERNANDEZ, L., ROSEMBLATT, M., AND HIDALGO, C. (1980) B&him Biophys. Acta 599,552-568. 20. MEISSNER, G. (1984) J. Biol. Chem. 259,2365-2374. 21. FABIATO, A., AND FABIATO, F. (1979) J. Physiol. (Paris) 75,463-505. 22. BRANDT, N. R., CASWELL, A. H., AND BRUNSCHWIG, J.-P. (1980) J. Biol Chem. 255,6290-6298. 23. HIDALGO, C., PARRA, C., RIQUELME, G., AND JAIMOVICH, E. (1986) Biochim. Biophys. Acta 855, 79-88.

AND

LOUIS

24. LAU, Y. H., CASWELL, A. H., BRUNSCHWIG, J.-P., BAERWALD, R. J., AND GARCIA, M. (1979) J. BioL Chem. 254,540-546. 25. SUMNICHT, G. E., AND SABBADINI, R. A. (1982) Arch. B&hem. Biophys. 215,628-637. 26. SABBADINI, R. A., AND OKAMOTO, V. R. (1983) Arch. Biochem. Biophys. 223,107-119. 27. JAIMOVICH, E., DONOSO, P., LIBERONA, J. L., AND HIDALGO, C. (1986) B&him. Biophys. Acta 855, 89-98. 28. LAU, Y. H., CASWELL, A. H., GARCIA, M., AND LETTELIER, L. (1979) J Gen Physiol. 74,335-349. 29. BRANDT, N. R., KAWAMOTO, R. M., AND CASWELL, A. H. (1985) J. Recept. Res. 5,155-170. 30. MADEIRA, V. M. C. (1982) Cell Calcium 3,67-79. 31. HIDALGO, C., GONZALEZ, M. E., AND GARCIA, A. M. (1986) Biochim. Biophys. Acta 854,279-286. 32. CAMPBELL, K. P., KNUDSON, C. M., IMAGAWA, T., LEUNG, A. T., SUTKO, J. L., KAHL, S. D., RAAB, C. R., AND MADSON, L. (1987) J. Biol. Chem. 262, 6460-6463. 33. PESSAH, I. N., WATERHOUSE, A. L., AND CASIDA, J. E. (1985) B&hem. Biophys. Res. Commun 128,449-456. 34. PESSAH, I. N., STAMBUK, R. A., AND CASIDA, J. E. (1987) Mol. Pharmacol. 31,232-238. 35. MEISSNER, G. (1986) J. Biol. Chem. 261,6300-6306. 36. IMAGAWA, T., SMITH, J. S., CORONADO, R., AND CAMPBELL, K. P. (1987) J. Biol. Chem. 262, 16636-16643. 37. NEWBOLD, R. P., AND TUME, R. K. (1981) J. Food Sci 46,1327-1332. 38. MEISSNER, G. (1975) B&him. Biophys. Acta 389, 51-68. 39. LOUIS, C. F., NASH-ADLER, P. A., FUDYMA, G., SHIGEKAWA, M., AKOWITZ, A., AND KATZ, A. M. (1980) Eur. .I B&hem. lll,l-9. 40. MITCHELL, G., AND HEFFRON, J. J. A. (1982) Adv. Food. Res. 28,167-230. 41. GREASER, M. L., CASSENS, R. G., HOEKSTRA, W. G., AND BRISKEY, E. J. (1969) J. Food Sci. 34, 633-637. 42. ROCK, E., AND KOZAK-REISS, G. (1987) Arch Biothem. Biophys. 256,703-707. 43. LOPEZ, J. R., DERSHWITZ, M., SANCHEZ, V., AND SRETER, F. A. (1988) Biophys. J. 53,609a. 44. CARONI, P., AND CARAFOLI, E. (1980) Nature &ondwn) 283,765-767. 45. MICKELSON, J. R., BEAUDRY, T. M., AND LOUIS, C. F. (1985) Arch. Biochem. Biophys. 242,127136. 46. KLIP, A., BRITT, B. A., ELLIOTT, M. E., WALKER, D., RAMLAL, T., AND PEGG, W. (1986) B&hem Cell Biol. 64,1181-1189.