Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

BlOCHEMlCAL

MEDICINE

AND

METABOLIC

BIOLOGY

38, 355-365

(1987)

Erythrocyte Membrane ATPase and Calcium Pumping Activities in Porcine, Malignant Hyperthermia HEMANT

S. THATTE,*

JAMES R. MICKELSON,? CHARLES F. Lourst

PAUL B. ADDIS,+ AND

*Dik*ision of Hematology, Department of Medicine. Stanford Uniivrsity School of Medicine. Stanford, California 94305, and PDepartments of Veterinary Biolog.v, and SFood Science and Nutrition, lJni\sersity of Minnesota, St. Paul, Minnesota 55108 Received

April

13, 1987

Malignant hyperthermia (MH) is a genetically transmitted hypermetabolic myopathy, triggered in susceptible individuals by potent volatile anesthetics such as halothane (1) or by depolarizing muscle relaxants such as succinyl choline (2). In MH-susceptible (MHS) swine, MH can be triggered by stressors such as exertion (3), heat, anoxia (4), or mechanical agitation (5). Most experimental findings on human and porcine malignant hyperthemia suggest there is an abnormality in the regulation of intracellular Ca7+ in skeletal muscle (6). A number of previous studies have indicated a skeletal muscle surface membrane defect in MH (7-l 1). That membrane defects may be expressed in cells other than the symptomatic tissue has resulted in a number of studies of erythrocyte membranes in different disease states (12-15). These membranes can be obtained in a relatively noninvasive manner for biochemical studies. We have recently reported an increased erythrocyte osmotic fragility in MHS pigs (16), suggesting a possible generalized surface membrane defect in this syndrome. While there have been a number of previous studies of erythrocyte fragility in MH (17-22), there have been few studies of the erythrocyte membrane ATPase enzyme activities (23,24) and none on erythrocyte calcium transport in MH. In the present study we report on the Mg*+-, (Na+, Kf)-, Ca’+-, and calmodulin-stimulable Ca’+-ATPase activities of MHS erythrocytes (25) and describe some of the kinetic parameters associated with these enzyme activities. In addition, the calcium transporting ability of calcium-loaded intact MHS and normal erythrocytes are compared. MATERIALS

The Pietrain and Yorkshire pigs used in this study were obtained from the University of Minnesota Experimental Farm. All Pietrain swine were classified as MHS as they developed extensor muscle rigidity when challenged with 3-5% halothane anesthesia for 3-4 min (26). Yorkshire swine were classified as normal 355 0085-4505187

$3.00

Copyright 0 1987 by Academic Press. Inc. All rights of reproduction in any form reserved.

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or malignant-hyperthermia negative as no adverse reaction developed even after 4 min of anesthesia. All the reagents used in this work were either of analytical grade or of the highest purity available. Calmodulin was prepared from bovine testes by the method of Gopalakrishna and Anderson (27). Water was deionized and distilled from glass. METHODS Collection ofblood. Blood was drawn from the cranial vena cava into a syringe containing acid citrate dextrose (ACD) anticoagulant, consisting of 89.6 mM sodium citrate, 141 mM dextrose, 15.6 mM citric acid, and 16.6 mM sodium phosphate buffer (pH 5.6), in a ratio of 10 vol of blood to 1.4 vol of ACD. The blood was stored at 0-4°C and was used within l-4 hr after collection. Isolation of erythrocyte ghost membranes. Blood was transferred to plastic centrifuge tubes and centrifuged at 4000g for 6 min at room temperature. The plasma and the buffy coat were aspirated and removed. Packed cells were washed three times with 0.16 M NaCl, and pH was adjusted to 7.4 with Tris (4”(Z), removing any remaining buffy coat at each step. Following the general procedure of Dodge et al. (28), erythrocytes were hemolyzed at 0-4°C in 20 mM imidazole, 1 mM EDTA (pH 7.4). The hemolysis buffer was added rapidly in a ratio of 14 vol of buffer to 1 vol of packed, washed erythrocytes. The resulting hemolysate was centrifuged at 48,000g for 30 min at 4°C and the supernate was discarded. The resulting pellet was resuspended in 20 mM imidazole, 1 mM EDTA (pH 7.4) and recentrifuged at 48,000g for 30 min at 4°C. This was repeated once or twice more such that the final pellet was white in appearance. Following each centrifugation step, the supernatant fluid was aspirated, and the tubes were gently rotated to allow the loosely packed ghost membranes to slide off the small button of unlysed cells and cellular debris. The pellet button was then carefully removed, and after the final centrifugation step, membranes were resuspended in a minimum volume of buffer. The membranes (approximately 4-6 mg protein/ml) were frozen in liquid nitrogen and stored at -70°C. Protein content of the membrane was determined by the method of Lowry et al. (29), using bovine serum albumin as standard. ATPase assay. Total (Na+, K+)-ATPase activity was assayed in 100 mM NaCl, 10 mM KCl, 1 mM EGTA, and 40 m histidine buffer (pH 7.4) at 37°C. Ouabainsensitive (Na+, K+)-ATPase activity was defined as that activity that was inhibited by 1 mM ouabain. Mg*+-ATPase activity was assayed in 120 mM KCl, 1 mM EGTA, 40 mM histidine buffer (pH 7.4) at 37°C. Ca*+-ATPase activity was assayed in 120 mM KCl, 01.1 mM CaCl,, 40 mM histidine (pH 7.4) and was defined as the difference between this activity and the Mg*+-ATPase activity. Calmodulinstimulable Ca*+-ATPase activity was defined as the ATPase activity in the presence of 0.1 ,UM calmodulin plus CaCl,, minus that in the presence of 0.1 PM calmodulin plus 1 mM EGTA. In experiments where Ca*’ was varied, Ca-EGTA buffers were used in the range 0.1-5 PM Ca*+, while Ca-nitrilotriacetic acid buffers were used in the range 5-760 PM Ca*+ (30).

MALIGNANT

HYPERTHERMIA

Ca-ATPase

357

ATPase assay mixtures, containing 0.15-0.25 mg protein/ml, were preincubated at 37°C for 3 min. Reactions were initiated with 5 mM Mg-ATP and were stopped at appropriate times by the addition of an equal volume of 2% (w/v) sodium dodecyl sulfate. Inorganic phosphate was estimated by the method of Rockstein and Herron (3 1). Calcium loading of intact erythrocytes and the determination of calcium efj‘lux. Red blood cells were loaded with 45Ca following the general method of Sarkadi et al. (32). In brief, the washed cells were suspended at a final hematocrit of 20% in ice-cold 0.16 M NaCl, 1 mM CaCl, (containing 2.5 &i/ml 45Ca), 5 mM ribose, 20 mM histidine (pH 7.4). A23187 (10 PM final concentration) was then added, after which the cells were placed in a 37°C water bath and allowed to incubate for 10 min, with frequent gentle mixing. Following this calcium-loading step, the A23187 was removed by washing the cells three times with ice-cold 0.16 M KCl, 20 mM histidine (pH 7.4), 0.1% bovine serum albumin (fatty acid free). The cells were then transferred to a calcium efflux medium containing icecold 0.16 M KCl, 5 mM ribose. 20 mM histidine (pH 7.4) at a final hematocrit of 20%. Calcium efflux was initiated by transfer to a 37°C water bath, and a total of six samples were taken at 1-min intervals for the determination of extracellular 4sCa. Rapid centrifugation through an oil layer was utilized to separate the cells from the extracellular media. The rate of erythrocyte calcium efflux under these conditions was linear with time for the 5-min experiment; thus the rate of calcium pumping could be determined by linear regression analysis. Control experiments demonstrated that the rate of calcium efflux was not affected by the inclusion of 1 mrvr CaCl, or 2 mM EGTA in the calcium efflux medium. Furthermore, influx of 45Ca did not occur during the time course of calcium efflux in cells which had been treated with A23187 and nonradioactive CaClz during the calcium loading step. Therefore the calcium efflux values reported in this study are not mediated in part by any A23187 remaining associated with the erythrocyte membranes following washing in serum albumin. Analysis of the data. The experimental data in this work were analyzed using Student’s t test. Values for the K, for Ca*’ and the V,,, of the erythrocyte ghost Ca”-ATPase activity and the calmodulin-stimulable Ca’+-ATPase activity were obtained from double reciprocal plots, fitted using linear regression leastsquares analysis. RESULTS ATPase Activities of MHS and Normal Erythrocytes Because of the variability of reported values for both Ca’+- and calmodulinstimulable Ca*+-ATPase activities of erythrocyte ghosts prepared by different procedures (33-40), it was first necessary to develop an isolation procedure which optimized these activities in pig erythrocyte ghosts. Preliminary experiments were performed to compare the properties of erythrocyte ghosts obtained by use of the 20 mM imidazole, 1 mM EDTA (pH 7.4) hemolysis media, with hemolysis buffers containing either 20 mM sodium phosphate (pH 7.4) or 20 mM Tris-HCl (pH 7.4). While the Mg+-ATPase, (Na+, K+)-ATPase, and Ca’+-ATPase activities

358

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ET AL.

TABLE 1 Comparison of ATPase Activities in Normal and MHS Erythrocyte

Breed

n

Normal MHS

18 18

Mg*‘-ATPase 0.42 t 0.22 0.44 f 0.21

(Na+,K’)-ATPase 0.45 f 0.126 0.31 r 0.11

Ca”-ATPase 1.57 k 0.47 1.31 f 0.40

Ghosts” Calmodulinstimulable Ca*‘-ATPase 10.48 f 2.5 9.58 f 2.0

Note. Erythrocyte ghost ATPase activities determined as described under Materials and Methods. a Means ? SD. Expressed as pmole Pi per milligram protein per hour at 37°C. b P < 0.0005; significantly greater than MHS erythrocytes. ’ P < 0.05; significantly greater than MHS erythrocytes.

did not differ greatly between ghosts obtained from the three different preparation media, the calmodulin-stimulable Ca’+-ATPase activity was three- to five-fold greater in ghosts prepared in imidazole buffer compared with those prepared in either phosphate or Tris buffer (data not shown). We therefore concluded that the ghosts prepared in imidazole buffer were most appropriate for our study. The erythrocyte ghost Mg*+-ATPase activities of normal and MHS erythrocyte ghosts were essentially identical (0.42 + 0.22 versus 0.44 + 0.21 pmole PJmg protein/hr) (Table 1). However, both the (Na+, K+) - ATPase and Ca*+-ATPase activities of normal erythrocyte ghosts were significantly greater than those activities in MHS ghosts (Table 1). There was no significant difference in calmodulinstimulable Ca*‘-ATPase activities between normal and MHS erythrocyte ghosts (10.48 ? 2.5 versus 9.58 + 2.0 pmoles PJmg protein/hi-, respectively). With the Ca*+-ATPase medium defined under Methods (25), the actual ionized Ca*’ concentration was 22 PM. We considered it possible that the difference in Ca*+-ATPase activity between normal and MHS erythrocyte ghosts could be greater at other Ca*+ concentrations. Examination of Fig. 1 indicates that in both types of ghosts, Ca*’ -ATPase activity exhibits two distinct phases (39): the first in the range 1.8-40 PM Ca*+ (reflecting the high-affinity Ca*+-binding site), and the second in the range 45-150 PM Ca*+ (reflecting the low-affinity Ca*+binding site). This confirms previous reports that erythrocyte ghost Ca*+-ATPase exhibits two classes of Ca*+-binding sites (41-43). In both MHS and normal ghosts, Ca*+-ATPase activity was maximal at approximately 280 PM Ca*‘; higher concentrations of Ca” inhibited Ca*‘-ATPase activity (Fig. 1). The kinetic parameters for the Ca*’ sensitivity of Ca*+-ATPase activity (Table 2) indicate that the K,,, for the high-affinity Ca*+ -binding site in normal ghosts (4.27 PM Ca*‘) is greater than that of MHS erythrocyte ghosts (1.15 PM Ca*+). In addition, the Ca*‘-ATPase V,,,,, for the high-affinity Ca*+-binding site of normal erythrocyte ghosts (2.07 pmoles PJmg protein/hr) is greater than that of MHS erythrocyte ghosts (1.32 pmoles PJmg protein/hr). The Km for the lowaffinity Ca*+-binding site in normal erythrocyte ghosts (78.66 PM Ca*‘) is smaller than that of MHS ghosts (93.8 PM Ca*+), while the V,,, for the low-affinity Ca*+-binding site in normal erythrocyte ghosts (6.96 pmoles Pi/mg protein/hi-) is greater than that of MHS erythrocyte ghost Ca*+-ATPase activity (5.49 pmoles

MALIGNANT I

I

Ilrlrr~

I

I

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HYPERTHERMIA I

f

rlrtlll

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359

Ca-ATPase I

I I I1111

I

I

6-

I; 54 ‘0 c i : 0 % zr =i

0

li1lll

IO

I

lllll

100

Free

Calcium

Cone

1000

(JIM)

FIG. 1. The effect of ionized Ca” concentration on the erythrocyte ghost Ca”-ATPase activity. ATPase activities of normal (0) and MHS (0) were assayed as described under Materials and Methods. n = 7 for each set, with data expressed as means k SD, with SD lines drawn only in one direction.

TABLE 2 Kinetic Parameters of Erythrocyte Ghost Ca” - ATPase and Calmodulin-Stimulable Ca” - ATPase Activities K, k SD (PM Ca’+ ) Calcium binding site High-affinity calcium binding site Low-affinity calcium binding site High-affinity calcium binding site in the presence of calmodulin

Normal 4.27

2 0.75

78.7

2 7.2

0.62

k 0.27

V m.,x *

SD

@mole P,/mg protein/hr) MHS

Normal

1.15 k 0.29"

MHS

2.07

f 0.36

1.32 -+ 0.13"

3.6"

6.96

2 1.50

5.9

2 0.13h

0.71 5 0.13'

25.0

k 3.4

22.7

2 I.2

93.8

2

Note. Erythrocyte ghost ATPase activities determined as described under Materials and Methods. Kinetic parameters are derived from data in Figs. 1 and 2. ” P < 0.0005; significantly different than normal. ’ P < 0.025; significantly different than normal. ’ Not significantly different than normal.

360

THATTE

0.1

ET AL.

1.0 Free

Calcium

IO Cont.

(uM)

FIG. 2. The effect of ionized Ca’+ concentration on the erythrocyte ghost calmodulin-stimulable Ca’+-ATPase activity. The enzyme activities of normal (0) and MHS (0) were assayed as described under Materials and Methods. n = 8 for normal and 7 for MHS, with data expressed as means + SD, with SD lines drawn only in one direction.

PJmg protein/hr). These differences in K, and V,,, are statistically significant (Table 2). Calmodulin-Stimulable

Ca2+-ATPase Activity

between the two breeds

of MH and Normal Erythrocytes

When the Ca2+ dependency of the calmodulin-stimulable Ca2’-ATPase activity was examined (Fig. 2), activity increased in the range 0.25-0.85 PM Ca2+; Ca2’ concentrations greater than 1 PM inhibited this activity. That calmodulin converts the Ca2+-ATPase from a form with both low and high Ca” affinity sites to a form with a single high-affinity Ca2’ site is in agreement with previous reports (41-43). The calmodulin-stimulable Ca2+- ATPase activities of normal and MHS erythrocyte ghosts exhibit single high-affinity sites for Ca2+ (K, = 0.62 PM Ca2+ and 0.71 PM Ca2+, respectively) that are not significantly different. Furthermore, both breeds exhibit a similar V,,,,, (25 pmoles PJmg protein/hr for normal and 22.7 @moles Pi/mg protein/hr for MHS (Table 2)). Both MHS and normal erythrocyte ghost Ca2+ -ATPase activity was stimulated by exogenous calmodulin (Fig. 3, in which the Ca2+ concentration was 22 PM). The calmodulin concentration required for half-maximal stimulation of Ca*+ATPase activity in MHS erythrocyte ghosts (K, = 11.7 nM calmodulin) is approximately twice that for normal erythrocyte ghosts (K,,, = 5.3 nM calmodulin); this difference was highly significant (Table 3). However, the V,,,,, for the calmodulinstimulable Ca2+-ATPase activity of normal erythrocyte ghosts under these conditons (Vmax = 13.3 pmoles Pi/mg protein/hr) is similar to that of MHS ghosts (V,,,,, = 12.9 pmoles PJmg protein/hr) (Table 3). This V,,, for calmodulin-stimulable Ca2+-ATPase activity in Table 3 is less than the V,,, value for the same activity

MALIGNANT I

I

HYPERTHERMIA I

lllll

361

Ca-ATPase I

I

I

1

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lllll

12 -

01

1

1

I

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Colmodu

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FIG. 3. The effect of calmodulin concentration on erythrocyte ghost Ca”-ATPase activity. ATPase activities of normal (0) and MHS (0) were assayed as described under Materials and Methods. n = 5 for each set with data expressed as means 2 SD, with SD lines drawn only in one direction.

in Table 2 because the Ca*+ concentration inhibits calmodulin-stimulable Ca*+-ATPase V max value. Calcium

Pumping

Activity

in the former case is 22 PM, which activity (Fig. 2), resulting in a lower

of Intact MH and Normal

Erythrocytes

The abnormalities in MHS erythrocyte ghost Ca*+-ATPase activities could result in an altered calcium pumping activity of intact MHS erythrocytes. To examine this possibility calcium efflux from erythrocytes was determined using cells that had been 45Ca loaded in the presence of 1 mM CaC12 and 10 PM A23187 (32). Following calcium loading and removal of the A23187, the rate of appearance of 45Ca in the extracellular medium was used to determine the rate of calcium efflux catalyzed by the erythrocyte membrane calcium pump (32). Under these TABLE 3 Kinetic Parameters for the Calmodulin Dependence of the Calmodulin-Stimulable Activity of MH and Normal Erythrocyte Ghosts” Breed

n

Normal MHS

5 5

VlW @mole P,/mg protein/hr) 13.3 1? 2.5 12.9 k I.8

Ca’+ - ATPase K? (nM calmodulin) 5.3 k 1.5 II.7 2 I.9h

Note. Erythrocyte ghost ATPase activities determined as described under Materials and Methods. Kinetic parameters are derived from data in Fig. 3. 0 Means 2 SD. b P < 0.0005; significantly different than normal erythrocytes.

362

THATTE

ET AL.

conditions the rate of MHS erythrocyte calcium efflux (0.234 + 0.015 nmoles Ca/liter cells/mm at 37°C) was significantly greater than that of normal erythrocytes (0.154 ? 0.019 mmoles Ca/liter cells/min at 37°C) means + SE for duplicate determinations on eight different MHS and normal pigs; P < 0.01). DISCUSSION A number of studies have indicated that abnormalities exist in membranes of MHS tissues, suggesting that there may be a generalized membrane involvement in MH (for review, see Ref (6)). These alterations in membrane function may result in an abnormal MHS intracellular Ca*’ regulation (7-11). Recent studies investigating the role of the plasma membrane in MH have identified alterations in erythrocyte osmotic fragility (16) and skeletal muscle sarcolemmal enzyme activities (IO, 11). In the present paper we have investigated aspects of the erythrocyte membrane Ca*+-ATPase and have unequivocally demonstrated a marked alteration in some of the kinetic parameters of this enzyme in membrane preparations from MHS pigs. The Ca*‘-ATPase of erythrocyte ghosts has been shown to exist in two different states (41-43). One state exhibits a low affinity for Ca*+ and has a high maximum activity, while the other state exhibits a high affinity for Ca*’ and has a low maximum activity. Our data demonstrate that the Ca*’ sensitivity of Ca*+ATPase activity is modified in MHS erythrocyte ghosts (Fig. 1 and Table 2). Thus, in the absence of calmodulin, the K,,, for Ca*’ stimulation of Ca*+-ATPase activity is smaller for MHS than for normal erythrocyte ghosts when Ca*+ is bound to the high-affinity site (Table 2). In contrast, when Ca*’ is bound to the low-affinity site (also in the absence of calmodulin), the K,,, for Ca*+ stimulation of Ca*+-ATPase activity is greater for MHS than for normal erythrocyte ghosts (Table 3). In the presence of calmodulin there is now no difference in the K,,, for Ca*+ stimulation of the Ca*+-ATPase activity (Fig. 2 and Table 2). Irrespective of the binding site occupied by Ca*+ in the absence of calmodulin, the V,,,,, for Ca*+-ATPase activity was always greater in normal than in MHS erythrocyte ghosts, while in the presence of calmodulin the Vmax'swere similar (Tables 2 and 3). In addition, the calmodulin concentration required for half-maximal stimulation of erythrocyte ghost Ca*+ -ATPase activity was approximately two fold greater in MHS than in normal membranes. That the kinetic parameters K,,,and V,,,,,for the Ca*+ sensitivity of the calmodulinstimulable Ca*‘-ATPase activity of MHS and normal erythrocyte ghosts are so similar (Table 2) would indicate there are similar total amounts of the Ca*+ATPase enzyme in these two types of membrane. Thus, the differences in Km and Lax values in the absence of calmodulin (Table 2) would appear to be due to some alteration in the MHS Ca*+-ATPase enzyme or its membrane environment, rather than the result of altered enzyme content in MHS ghosts. That Mg*+ATPase activities are very similar in MHS and normal ghosts (Table 1) would indicate that, like the Ca*+-ATPase, Mg*+- ATPase content is similar in MHS and normal ghosts. The explanation for the increased (Na+ , K+)-ATPase activity of normal ghosts in comparison with MHS ghosts is not immediately clear (Table 1).

MALIGNANT

HYPERTHERMIA

Ca-ATPase

363

In the only previous study that describes erythrocyte ghost ATPase activities in MH, Mg’+-, and (Na+, Kf)-ATPase, activities were reported to be unaltered (18,23). However, the previous data (18,23) were derived from a study of human erythrocyte ghosts derived from just two individuals, whereas the present study used up to 18 MHS and normal swine. Interestingly, the CaZf-ATPase has been reported to be lOO-200% increased in erythrocyte ghosts isolated from two dogs diagnosed as MHS (24). However, in contrast with swine and human erythrocyte ghosts, ghosts from dogs are calmodulin insensitive, indicating that they may have additional mechanisms for regulating Ca” levels in their red blood cells (44). In human red blood cells, the total calmodulin concentration is thought to be in the range 2.5-7 PM (44), while the free Ca2+ concentration is in the range 0.01-0.25 PM (45). If similar concentrations occur in porcine red blood cells, then it is likely that the Ca2+-ATPase is in the calmodulin-stimulable form. This form exhibits similar kinetics in MHS and normal erythrocytes (Table 2). Thus, the red blood cell membrane calcium pump, which is a reflection of the Ca2+ATPase activity, might not demonstrate abnormalities between MHS and normal animals in vivo. This was confirmed experimentally using 45Ca - loaded intact erythrocytes from MHS and normal pigs. Under the conditions of our experiments, for as yet unknown reasons, the calcium pumping ability of the MHS cells was significantly greater than that of the normal cells. We would conclude that differences in the kinetic parameters of the erythrocyte membrane Ca’+-ATPase in MH provide evidence for a generalized membrane defect in this syndrome. However, these differences do not result in an altered erythrocyte Ca’+ regulation in MH. SUMMARY

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg’+-ATPase activities, the (Na+. K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca’+-ATPase activity exhibited low- and high-affinity Ca’+-binding sites in both types of erythrocyte ghost. While the K,,, for Ca’+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca’+-binding site, the reverse was true at the low-affinity Ca2+ -binding site. Irrespective of the type of calcium binding site occupied, the V,,,,, for normal erythrocyte ghost Ca’+ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca’+ or the V,,, of the Ca’+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are

364

THATTE

unlikely to result in an impaired cytosolic Ca2+ concentration.

ability

ET AL.

of MHS

erythrocytes

to regulate their

ACKNOWLEDGMENTS The authors thank Dr. Bill Rempel for the invaluable supply of experimental animals, Dr. Esther Gahant for helpful discussions, and Janine White, Peggy Hogan, and Kay Johnson for excellent laboratory assistance.

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12. 13. 14. 15. 16.

17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

897, 364 (1987).

Huninger, R. N.. and Cheung, H. C., C/in. Chim. Acta 156, 165 (1986). Dunn, M. J., Burghes. A. H., and Dubowitz, V., Biochem. J. 201, 445 (1982). Tsuchiya, Y., Sugita, H.. and Kuroiwa, Y., Biochem. Med. 30, 271 (1983). Muallem, S.. Miner, C., and Seymour, C. A., Biochim. Biophys. Acta 819, 143 (1985). O’Brien, P. J., Rooney, M. T., Reik, T. R., Thatte, H. S., Rempel. W. E., Addis, P. B., and Louis, C. F., Amer. J. Vet. Res. 46, 1451 (1985). Harrison, G. G., and Verburg, C., Brit. J. Anaesthesiol. 45, 131 (1973). Godin, D. V., Herring, F. G., MacLeod, P. J. M., J. Med. 12, 35 (1981). Lampo, P., Rev. Agric. (Brussels) 31, 843 (1978). Kursa, J., and Kroupova, V., Res. Vet. Sci. 20, 97 (1976). Heffron, J. J. A., and Mitchell, G., Brit. J. Anaesthesiol. 53, 499 (1979). King, W. A., Ollivier, L., and Basrur, P. K., Ann. Genet. Sel. Anim. 8, 537 (1976). Godin, D. V., Bridges, M. A., and MacLeod, P. J. M., J. Med. 10, 287 (1979). O’Brien, P. J., Forsyth, G. W., Olexson. D. W., Thatte, H. S., and Addis, P. B. Canad. J. Camp. Med. 48, 381-389 (1984). Carafoli, E., and Zurini, M., Biochim. Biophys, Acta 683, 279 (1982). Harrison, G. G., Biebuyck, J. F., Terblanche, J., Dent, D. M., Hickman, R., and Saunders, J., Brit.

Med.

J. 3, 594 (1968).

27. Gopalakrishna, R., and Anderson, W. B., Biochem. Biophys. Res. Commun. 104, 830 (1982). 28. Dodge, J. J., Mitchell, C., and Hanahan, D. J., Arch. Biochem. Biophys. 100, 119 (1963). 29. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.. J. Biol. Chem. 193, 265 (1951).

30. 31. 32. 33. 34. 35. 36. 37.

Fabiato, A., and Fabiato, F., J. Physiol. (Paris) 75, 463 (1979). Rockstein, M., and Hen-on, P. W., Anal. Chem. 23, 1500 (1951). Sarkadi, B., Szasz, I., and Gardos, G., .I. Membr. Biol. 26, 357 (1976). Roufogalis, B. D., Canad. J. Physiol. Pharmacol. 57, 1331 (1979). Sarkadi, B., Biochim. Biophys. Acra 604, 159 (1980). Muallem, S., and Karlish, S. J. D., Biochim. Biophys,. Actn 597, 631 (1980). Quist, E. E., and Roufogalis, B. B., Arch. Biochem. Biophys. 168, 240 (1975). Schatzmann, H. J., and Vincenzi, F. F., .I. Physiol. 201, 369 (1969).

MALIGNANT 38. 39. 40. 41. 42. 43. 44. 45.

HYPERTHERMIA

Ca-ATPase

Dunham, E. T., and Glynn, I. M., J. Physiol. 156, 274 (1961). Farrance, M. L., and Vincenzi. F. F., Biochim. Biophys. ACM 471, 49 (1977). Thakar, J. H., Anal. Biochem. 144, 94 (1985). Scharff, 0.. Biochim. Biophys. AC&I 443, 206 (1976). Scharff, 0.. and Foder, B., Biochim. Biophys. Acta 483, 416 (1977). Scharff, O., and Foder, B., Biochim. Biophys. Acta 509, 67 (1978). Vincenci. F., Red Cell Ann Arbor Conf 5th, 363 (1981). Schatzmann. H. J.. Cum. Top. Mrmbr. Transp. 6, 125 (1975).

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