Characterization in rainbow trout
of myocardial
Na+-Ca2+ exchange
GLEN F. TIBBITS, KENNETH D. PHILIPSON, AND HARUYO KASHIHARA Cardiac Membrane Research Laboratory, Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1 S6, Canada; and Cardiovascular Research Laboratory, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024
Tibbits, Glen F., Kenneth D. Philipson, and Haruyo Kashihara. Characterization of myocardial Na’-Ca” exchange in rainbow trout. Am. J. Physiol. 262 (Cell Physiol. 31): C4ll-C417, 1992.-This study compared Na’-Ca”’ exchange from the hearts of rainbow trout with that from canines. In several respects, trout cardiac Na’-Ca2’ exchange is functionally similar to that from dogs and other mammals. Trout cardiac Na’-Ca” exchange is stimulated -200% after 30-min incubation with 10 pg/ml chymotrypsin at 21°C, similar to mammals. On the other hand, both the temperature and pH dependencies are strikingly different between the trout and canine myocardial Na+-Ca”+ exchange. While canine heart Na’-Ca”’ exchange exhibits a Q10 of >2 (similar to values observed in other mammals), that from trout is relatively insensitive to temperature with a Q10 of -1.2. The absolute rates of Na’-Ca” exchange in trout heart are four- to sixfold higher than that in mammals when measured at 7°C. Furthermore, the temperature insensitivity of trout myocardial Na’Ca” exchange is retained when the exchanger is reconstituted into an asolectin bilayer, suggesting that this property is intrinsic to the protein and not dependent on species differences in lipid bilayer composition. Trout Na+-Ca2+ exchange is not markedly stimulated by alkaline pH, in contrast to mammals, and this characteristic is also maintained after reconstitution. Western blots of trout cardiac sarcolemma run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis react with antibodies raised against the canine Na+-Ca2+ exchanger with a similar pattern of bands (70,120, and 160 kDa). Furthermore, a cDNA probe from canine Na’-Ca” exchanger hybridizes on Northern blots of trout heart mRNA to a 7-kb band, similar to that in mammals. Thus, while important functional differences in Na’-Ca” exchange exist between trout and mammalian hearts, the molecular basis is not yet known. proteinase; ence
messenger
ribonucleic
acid; temperature
depend-
myocardial contraction is dependent on a transsarcolemmal influx of calcium (17), which has been proposed by Fabiato (13) to trigger the release of a greater quantity of calcium from the sarcoplasmic reticulum (SR). Na+-Ca2+ exchange is the primary mechanism of Ca”+ efflux across the sarcolemma (SL) in the mammalian heart and contributes, therefore, to mechanical relaxation (4, 7). Furthermore, it has been suggested recently that the Na+-Ca”+ exchanger contributes to SR Ca’+ release (18). Thus it is well established in mammals that Na’-Ca2’ exchange is an integral component in the regulation of myocardial contractility (26). Much less is known about Na’-Ca2’ exchange and contractility in the hearts of lower vertebrates. Electron microscopy of a number of poikilotherm hearts, including the trout, demonstrates both a sparsity of SR (28) in comparison to the mammalian heart and an absence of IN MAMMALS,
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Copyright
transverse tubules. Furthermore, contraction in the amphibian (3) and teleost (12) ventricle appears to be relatively insensitive to ryanodine in doses known to block SR Ca2+release. Therefore, unless mitochondria contribute to myofilament Ca2+, an intracellular source of Ca2+ is of less significance in cardiac contraction in lower vertebrates. It should be noted, however, that in mammals a role for mitochondrial Ca2’ release in regulating contraction on a beat-to-beat basis has been dismissed based on a variety of arguments (8). Given these findings, it has been suggested that the magnitude of transsarcolemma1 Ca2+ transport in lower vertebrates is sufficient to support contraction (16, 30) and that Na’-Ca2’ exchange is the primary means of reducing cytosolic [Ca”‘] with each beat (29). Furthermore, active teleosts, such as rainbow trout and other Salmonidae, maintain reasonable cardiac outputs at temperatures (48°C) that are cardioplegic to mammals, demonstrating that Ca2’ transport systems are not dysfunctional in these species at low temperatures. We predicted, therefore, that trout Na+-Ca2+ exchange is comparatively more active than the mammalian exchanger under hypothermic conditions. As a consequence, this study sought to characterize the cardiac Na+-Ca2+ exchanger in SL from rainbow trout and compare it with that from mammals. METHODS Sarcolemmal isolation. Rainbow trout (Oncorhynchus mykiss) weighing 350-500 g were obtained from West Creek Trout Farm (Aldergrove, Canada) and maintained locally for a minimum of 2.5 wk at a temperature of +‘C. After the fish were killed with a sharp blow to the head, a ventral incision allowed the heart to be excised quickly. Typically, 12 trout ventricles were pooled, and each SL was isolated as described previously (30). SLs were isolated from the ventricles of mongrel dogs as detailed by Philipson and Ward (25). SL vesicles from both species were resuspended in loading medium (LM) which contained 140 mM NaCl and 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.4 at 21°C). Protein was determined in native sarcolemmal vesicles (NV) either by the Bradford (6) or Lowry (19) assays. Reconstitution. NV from both trout and canine hearts were reconstituted into asolectin membranes with modifications of a technique described previously (32). In brief, 33 ~1 of native SL (-3 mg/ml) were solubilized in 1 vol of 15 mM decylmaltoside and 140 mM NaCl. After spinning for 10 min at 178,000 g at room temperature in an Airfuge to remove nonsolubilized material, the supernatant was diluted with an equal volume of 140 mM NaCl and one-fourth volume of an asolectin mixture. The latter was produced by dissolving asolectin (50 mg/ml) in 7.5% Triton X-100, 100 mM MOPS-tris(hydroxymethyl)aminomethane (Tris) (pH 7.4 at 37”C), and 2.5 M NaCl and
0 1992 the American
Physiological
Society
c411
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MYOCARDIAL
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then sonicated for clarity. The resultant suspension was agitated in the presence of approximately one-fourth volume of BioBeads SM-2 for 20 min, and this step was repeated once to ensure removal of the detergents. After this treatment, the vesicles were diluted to 400 ~1 with LM and spun for 40 min in an Airfuge. The resultant pellet was carefully resuspended in 50 ~1 of LM. Na+-Ca” enchange. Sodium-dependent calcium uptake into NV was performed as described (24). In brief, 5 ,ul of Na’loaded sarcolemmal vesicles were suspended on the wall of a polystyrene tube containing 245 ~1 of uptake medium maintained at 21°C (unless indicated otherwise). The medium contained either 140 mM KC1 or NaCl, 2 &i 45Ca2+, 0.5 PM valinomycin, 20 PM 40Ca”‘, and, unless indicated otherwise, 10 mM MOPS (pH 7.0 at 21°C). In experiments in which the temperature dependence was to be determined, the vesicles were maintained at 4°C until suspended above the reaction media maintained at the desired temperature. In assays in which the extracellular pH (pH,) dependence was to be evaluated, 10 mM Tris maleate was used in lieu of MOPS and the pH was varied from 5.0 to 9.0. The uptake was initiated by vortex mixing and quenched at 2 s by the addition of 30 ~1 of a solution containing 140 mM KC1 and 1 mM LaC&. A 2209~1 aliquot was filtered (0.45 pm Millipore), and the filters were washed with 3 ml of a rinsing solution (containing 140 mM KC1 and 1 mM LaClJ twice. The filters were dried and then counted. The uptake by vesicles diluted into NaCl represented blanks and was subtracted for all data points. This allows for the correction of 4FiCa’2+ that was bound superficially to the sarcolemma or had permeated the vesicle by some pathway other than Na’-Ca”’ exchange. In all cases, this value was no greater than 10% of that obtained with K’ dilution. The Na+-dependent Ca”’ uptake into reconstituted vesicles (RV) was performed in a similar fashion as that in native vesicles except that 1) 3 ~1 of RV were used, 2) the reaction was quenched with 140 mM KC1 and 10 mM ethylene glycolbis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 3) the filters had a pore size of 0.22 pm, and 4) the filters were rinsed with 140 mM KC1 and 1 mM EGTA maintained at 4°C. Protein was determined in RV by the method of Wang and Smith (33). Chymotrypsin treatment. NV from both trout and canine hearts were incubated with 10 pg/ml chymotrypsin for 30 min at 21°C. After this period of time, an aliquot of SL was mixed with the sample buffer in preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described, and the remainder was immediately used for Na’dependent Ca” uptake experiments. The latter was determined at 21°C and pH, 7.0. SDS-PAGE and Western blots. SDS-PAGE was performed on NV from trout and canine hearts as described (23) in 7.5% polyacrylamide. The proteins were transferred to nitrocellulose membranes electrophoretically and reacted with antibodies raised against the purified canine exchanger (23). Isolation of mRNA and Northern blots. Isolation of mRNA from both trout and canine heart followed the procedure of Chomczynski and Sacchi (10). Briefly, trout hearts were excised and frozen immediately in ethanol chilled with dry ice and then maintained at -70°C. About 2 g of frozen hearts were added to 30 ml of a denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) in a Waring blender and immediately homogenized for two bursts of 30 s each. The final pellet was dissolved in 0.5 ml diethyl pyrocarbonate (DEPC)-treated water. A l-p1 aliquot of the total RNA was analyzed spectrophotometrically and the remainder was used to isolate poly(A)+ RNA by oligo(dT) chromatography as described by Aviv and Leder (1). A l-p1 aliquot of the poly(A)+ RNA was also analyzed spectrophoto-
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metrically and then stored in liquid N, until used. RNA was denatured by heating for 5 min at 65°C in a solution containing formaldehyde and formamide. About 5 pug of denatured RNA were added to each well of a 1% agarose, 2.2 M formaldehyde gel. After separation, the RNA was transferred passively to a nitrocellulose membrane. A ‘31”C after reconstitution compared with the native membrane. The pH dependence of the Na’-Ca” exchanger from both species in native sarcolemmal vesicles is shown in Fig. 4. In the canine heart, there is an approximate 270% stimulation of Na’-Ca2’ exchange by increasing the pH from 7.0 to 9.0. No stimulation was observed in the trout heart Na’-Ca2’ exchange. A qualitatively similar result was obtained with the Na’-Ca2+ exchangers from both species after being reconstituted into asolectin vesicles (Fig. 5). Chymotrypsin, at a concentration of 10 pg/ml, stimulated the trout and canine Na+-dependent Ca”+ uptake in NV by -194 and 200%, respectively, as shown in Fig. 6. In both species, it was noted that this stimulation was associated with a more intense 70-kDa band and reduced intensity of the 120-kDa band in Western blots (data not shown). A Western blot using antibodies raised against the purified canine Na+-Ca2+ exchanger and SL from trout and canine hearts is shown in Fig. 7. The pattern of banding was similar in both species, with bands consistently appearing at 70, 120, and 160 kDa. The total RNA yield was 0.88 and 2.25 pg/mg wet wt in the canine and trout hearts, respectively. The 260/280
Trout Canin
I
1
I
7
21
35
Temp
(lOOO/‘K)
Fig. 2. Arrhenius plot of the data shown in Fig. 1. Na+-dependent Ca2+ uptake was normalized for the rate at 37°C. Derived Qlo values of Na’Ca”’ exchange were 1.2 in the trout (7-37°C) and 2.0 (Zl-37°C) and 5.5 (7-21°C) in the canine heart. NV, native sarcolemmal vesicles.
c413
TROUT
(OC)
Fig. 3. Initial rates of Na+-dependent Ca”’ uptake in sarcolemmal vesicles (RV) as a function of temperature, for the rate at 21°C. [Ca’+], was 20 PM, n = 4 different for each group, and the reaction time was 2 s. RV were described in METHODS and maintained at 4°C until the initiated. 100% equals 8.4 and 8.6 nmol. mg protein-’ l s-l canine RV, respectively.
reconstituted normalized preparations prepared as reaction was in trout and
ratio was 1.60 and 1.74 for canine and trout RNA, respectively. In the agarose gels of poly(A)+ RNA, the 28s ribosomal RNA from the trout heart (-4.0 kb) always migrated farther than that from the canine heart (-4.4 kb). The canine heart 28s rRNA in all cases migrated the same distance as that from rat heart, brain, and adrenals, as well as rabbit heart and dog pancreas (data not shown). In the Northern blot, the cDNA probe from the canine heart Na’-Ca2’ exchanger hybridized nonspecifically to the 28s rRNA from both trout and canine heart. Although faint in the autoradiograph of the Northern blot shown in Fig. 8, the cDNA probe from canine heart Na+-Ca”+ exchanger routinely hybridized to a -7-kb band of trout mRNA, similar to mammals. DISCUSSION
The temperature dependence of Na+-Ca2+ exchange in the canine heart was similar to that observed by others using a variety of mammals, including dogs (5). Bersohn et al. (5) recently demonstrated that the amphibian heart Na+-Ca2+ exchange was less temperature dependent than that in mammals. The Arrhenius plot for Na+-Ca2+ exchange showed clear breaks at -22°C for both mammalian and amphibian hearts. In the present study, the teleost cardiac Na’-Ca”’ exchange shows even less temperature dependence than that of the frog and no obvious breakpoint in the Arrhenius plot. These properties were maintained in asolectin vesicles in all species. Therefore,
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c414
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300
0 l
Trout Canine
NA+-CA’+
EXCHANGE
NV NV
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TROUT
0
Trout
m
Canine
50 -CT 0
+CT
Fig. 6. Initial rates of Na+-dependent Ca2+ uptake in NV before (-CT) and after (+CT) chymotrypsin treatment as described in METHODS. [Ca2+10 was 20 PM, n = 3 different preparations for each group; temperature was 21”C, and the reaction time was 2 s.
Fig. 4. Initial rates of Na+-dependent Ca*+ uptake in NV as a function of pH, normalized for the rate at pH 7.0. 100% equals 3.1 and 3.4 nmol. mg protein-‘.s-’ in trout and canine NV, respectively. [Ca2+10 was 20 PM, n = 4 different preparations for each group; temperature was 21”C, and the reaction time was 2 s.
-160 -120 200 0 c z a g 150
l
Trout Canine
RV RV
- 70
be
Tr
~,
,
0 5
7
Ca
Fig. 7. Western blot of native sarcolemma from trout (Ti; 23.4 pg) and canine (Ca: 9.7 ue) hearts run on 7.5% SDS-PAGE. Primarv I antibodies w&e raised & rabbits against purified canine cardiac Na+Ca” exchanger as described (23). Second antibody was goat anti-rabbit conjugated to horseradish peroxidase. Numbers on right refer to relative molecular mass (in kDa).
9
PH Fig. 5. Initial rates of Na+-dependent Ca*+ uptake in RV as a function of pH, normalized for the rate at pH 7.0. [Ca2+10 was 20 PM, n = 4 different preparations for each group; temperature was 21”C, and the reaction time was 2 s.
the species differences in temperature dependencies of Na+-Ca2+ exchange are apparently independent of possible differences in bilayer composition and must reflect differences in protein structure. In both Salmonidae (20) and mammals (5), the Michaelis constant for Ca2+
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MYOCARDIAL
NA+-CA’+
EXCHANGE
4.4
Fig. 8. Autoradiograph of Northern blots of agarose gels of 5 pg of mRNA isolated from total RNA by oligo(dT) affinity chromatography from trout (Tr) and canine (Ca) hearts. The 32P-labeled probe was a 200-bp segment of clone A4 described by Nicoll et al. (21) from cDNA of the canine heart Na+-Ca2+ exchanger. Hybridization was performed as described in METHODS. Numbers on right refer to size of mRNA (in kb), and probe hybridizes to -7-kb mRNA segment in both trout and canine hearts.
[K,(Ca)] of Na+-Ca2+ exchange is unaffected by temperature, and thus the observed effects of temperature are due strictly to changes in maximum velocity (If,,,). The species variation in temperature dependence is an important physiological difference because cold-adapted Salmonidae maintain relatively high cardiac outputs at temperatures as low as 4°C. If Na+-Ca2+ exchange is responsible for the majority of cytosolic Ca2+ removal with each beat, as has been suggested (30), then this represents an important evolutionary adaptation. Na+Ca2+ exchange from crayfish skeletal muscle (27) has a reasonably similar temperature profile as that for trout myocardial Na+-Ca2’ exchange. In both trout cardiac (20) and crayfish skeletal muscle Na+-Ca2+ exchange (27), there was an obvious decline in activity when the membranes were maintained at >25”C for more than a brief period. Thus, in the present study, the temperature profile was determined with an exposure to the reaction temperature of no more than 2 s, and also the chymotrypsin proteolysis was carried out at 21°C. While a teleological explanation for the differences between poikilo- and homeotherms is apparent, a mechanistic interpretation is not. On the basis of the temperature profiles of cardiac Na+-Ca2+ exchange activity, there appears to be at least three different classes (mammalian, amphibian, and teleost) of exchangers that presumably reflect differences in the primary structure of the protein. Similar temperature-dependent species differences have been observed for the Ca2+ sensitivity of the cardiac contractile element (14). It should be noted, however, that trout p-nitrophenyl phosphatase (K+-pNPPase), a sarcolemmal protein, exhibits a temperature dependence radically different from that of the Na+-Ca2+ exchanger and not unlike that of the mammalian exchanger (G. F. Tibbits, unpublished observations). The lack of stimulation of trout cardiac Na+-Ca2+ exchange by alkaline pH was unexpected. The stimula-
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c415
tion of canine cardiac Naf-Ca2+ exchange by alkaline pH observed in this study is similar to that observed in previous studies (22) on mammalian hearts. Similarly, this stimulation is observed when the exchanger is reconstituted into asolectin (32), indicating that this property is intrinsic to the protein itself. The alkaline-induced increase in Na+-Ca2+ exchange has also been observed in other mammalian tissues (26) as well as in invertebrates including squid giant axons (2, 11) and crayfish skeletal muscle (27). The stimulation of the cardiac exchanger is the result of lowering the &(Ca) of the exchanger at alkaline pH (22). It was postulated (22) that a histidine residue near a Ca2+ binding site on the exchanger becomes deprotonated at higher pH and changes the conformation of the Ca*+ binding site. However, other possible hypotheses remain viable and therefore a mechanistic explanation of the species difference is not possible. A teleological explanation for the lack of stimulation of trout Na+-Ca2+ exchange by high pH is not apparent. It should be noted, however, that in poikilotherms in which body temperatures vary with ambient conditions, both pH, and intracellular pH also fluctuate in a predictable manner, -O.O17/“C rise in temperature (15). This relationship is known to covary with the temperature dependencies of the pK of imidazole and the neutral pH of water (15). Whereas the “normal” pH, at 37°C is 7.4, it is likely to be 7.8 at 4°C. In the present study, pH, was maintained at 7.0 while temperature was allowed to vary from 7 to 37°C to isolate the independent effects of pH and temperature. The differences in the temperature and pH dependencies of Na+-Ca2+ exchange between the trout and dog hearts cannot be attributed to differences in the sarcolemma1 isolation procedures. Properties of the canine Na+-Ca2+ exchange described in this study are qualitatively similar to those from a variety of mammalian species using different SL isolation procedures (26). Furthermore, an SL isolation procedure for rat hearts that was identical to that used for trout in the present study yielded qualitatively similar results to that from the canine heart (G. F. Tibbits, unpublished observations). Similarly, it is unlikely that the species differences in pH and temperature dependencies of Na+-Ca2+ uptake observed in the reconstituted vesicles are due to contaminating endogenous phospholipids. This is based on the facts that 1) the ratio of exogenous to endogenous phospholipid is in excess of 12:l and 2) exchange activity in the reconstituted vesicles has an absolute requirement for specific exogenous lipids (32). From studies of this type, there is no evidence that the endogenous lipids form an annulus around the protein that is difficult to replace with exogenous lipids during reconstitution. As shown in Fig. 6, the stimulation of trout myocardial Na+-Ca2+ exchange by limited chymotrypsin proteolysis is similar to that observed in mammals in both the present study and others (24). Both the higher and lower vertebrates can be distinguished from the Na+-Ca2+ exchanger of the invertebrate Artemia which is insensitive to limited proteolysis (9). A Western blot of both canine and trout SL run on SDS-PAGE and reacted with polyclonal antibodies raised in rabbits to Na+-Ca2+ exchanger purified from
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C416
MYOCARDIAL
NATA’)+
EXCHANGE
dog heart is shown in Fig. 7. The specificity of the polyclonal antibodies for the Na+-Ca”+ exchange protein has been demonstrated with two different monoclonal antibodies (Ref. 31, and Frank et al., unpublished observations). Because this reaction was not as pronounced in trout, as compared with dog, typically 2.5 times as much protein was used per lane for trout. This is likely due to a lower antigenicity of trout Na+-Ca2+ exchanger compared with dog but is also possibly due to lower exchanger density in the SL preparations. The K+-pNPPase activhigher compared with ity in canine SL was -25fold trout SL and the amount of purification was also comparably higher. The pattern of banding on the Western blot was similar in both species. Recently, the canine Na’-Ca’+ exchange cDNA was cloned and sequenced, and the deduced molecular mass is 108 kDa (21). The 120-kDa band may be the consequence of posttranslational modification such as glycosylation. Regardless, the native molecular weight of the Na+-Ca*’ exchanger appears to be similar in the two species. In agarose gels of poly(A)+ RNA, the 28s rRNA observable at 4.4 kb in mammals always migrated further in trout for reasons that are unknown. Northern blots of these gels are shown in Fig. 8. A cDNA probe from the canine cardiac Na+-Ca*+ exchanger hybridizes to trout mRNA with a band at -7 kb similar to that in mammals. This indicates that there is some homology between Na+Ca’)+ exchanger cDNAs from the two species, and the transcripts are approximately the same size. These experiments, in general, show a high degree of similarity in the properties of the cardiac Na’-Ca*’ exchangers from mammals and teleosts. These similarities include K,(Ca) (29), stimulation by both valinomycin (29) and limited chymotrypsin proteolysis, apparent molecular weight of the protein, and size of transcript. The most striking difference between the two species is the temperature dependence that is likely due to differences in the protein amino acid sequence. Because the mammalian Na+-Ca”+ exchanger antibodies cross-react with trout Na+-Ca”+ exchanger and because mammalian Na+Ca’+ exchanger cDNA hybridizes to trout Na’-Ca*’ exchanger mRNA, there is significant homology between the two species. A mechanistic interpretation of the different temperature profiles will require a more detailed knowledge of the structure of the myocardial Na+-Ca”+ exchangers. We gratefully acknowledge the generous support of the National Sciences and Engineering Research Council (Canada), the British Columbia Health Research Foundation (to G. F. Tibbits), and the National Institutes of Health (to K. D. Philipson). The contributions of Drs. Zhaoping Li, John Keen, Deb Nicoll, and Marion Thomas are appreciated. Address for reprint requests: G. F. Tibbits, Cardiac Membrane Research Laboratory, Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A lS6, Canada. Received
20 June
1991; accepted
in final
form
28 August
1991.
REFERENCES 1. Aviv, H., and P. Leder. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc. Natl. Acad. Sci. USA 69: 1408-1412, 1972. 2. Baker, P. F., M. P. Blaustein, A. L. Hodgkin, and R. A. Steinhardt. The influence of calcium on sodium efflux in squid
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axons. J. Physiol. Land. 200: 431-458, 1969. 3. Bers, D. M. Ca influx and sarcoplasmic reticulum Ca release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248 (Heart Circ. Physiol. 17): H366-H381, 1985. 4. Bers, D. M., and J. H. B. Bridge. Relaxation of rabbit ventricular muscle by Na-Ca exchange and sarcoplasmic reticulum calcium pump. Circ. Res. 65: 334-342, 1989. 5. Bersohn, M. M., R. Vemuri, D. S. Schuil, R. S. Weiss, and K. D. Philipson. Effect of temperature on Na’-Ca” exchange in sarcolemma from mammalian and amphibian hearts. Biochim. Biophys. Acta 1062: 19-23, 1991. 6. Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal. Biochem. 72: 248-254, 1976. 7. Bridge, J. H. B., K. W. Spitzer, and P. R. Ershler. Relaxation of isolated ventricular cardiomyocytes by a voltage-dependent process. Science Wash. DC 241: 823-825, 1988. 8. Carafoli, E. Intracellular calcium homeostasis. Annu. Reu. Biochem. 56: 395-433, 1987. 9. Cheon, J., and J. P. Reeves. Sodium-calcium exchange in membrane vesicles from Artemia. Arch. Biochem. Biophys. 267: 736741, 1988. 10. Chomczynski, P., and N. Sacchi. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 11. DiPolo, R. The sodium-calcium exchange in intact cells. In: Sodium-calcium Exchange, edited by T. J. A. Allen, D. Noble, and H. Reuter. Oxford, UK: Oxford Univ. Press, 1989, p. 5-26. 12. Driedzic, W. R., and H. Gesser. Differences in force-frequency relationships and calcium dependency between elasmobranch and teleost hearts. J. Exp. Biol. 140: 227-241, 1988. 13. Fabiato, A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J. Physiol. 245 (Cell Physiol. 14): ClC14,1983. 14. Harrison, S. M., and D. M. Bers. Temperature dependence of myofilament Ca sensitivity of rat, guinea pig and frog ventricular muscle. Am. J. Physiol. 258 (Cell Physiol. 27): C274-C281, 1990. 15. Hochachka, P. W., and G. N. Somero. Biochemical Adaptation. Princeton, NJ: Princeton Univ. Press, 1984. 16. Klitzner, T., and M. Morad. Excitation-contraction coupling in frog ventricle: possible Ca”’ transport mechanisms. Pfluegers Arch. 398: 274-283,1983. 17. Langer, G. A. The role of calcium in the control of myocardial contractility: an update. J. Mol. Cell. Cardiol. 12: 231-239, 1980. 18. Leblanc, N., and J. R. Hume. Sodium-current-induced release of calcium from cardiac sarcoplasmic reticulum. Science Wash. DC 248: 372-376,199O. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951. 20. McKnight, K. J., H. Kashihara, and G. F. Tibbits. Na’-Ca” exchange in sarcolemma from a salmonid heart: seasonal variations (Abstract). Can. Fed. Biol. Sot. Proc. 32: 150, 1989. 21. Nicoll, D. A., S. Longoni, and K. D. Philipson. Molecular cloning and functional expression of the cardiac sarcolemmal Na’Ca” exchanger. Science Wash. DC 250: 562-565, 1990. 22. Philipson, K. D., M. M. Bersohn, and A. Y. Nishimoto. Effects of pH in Na’-Ca” exchange in canine cardiac sarcolemmal vesicles. Circ. Res. 50: 287-293, 1982. 23. Philipson, K. D, S. Longoni, and R. Ward. Purification of the cardiac Na’-Ca”’ exchange protein. Biochim. Biophys. Acta 945: 298-306,1988. 24. Philipson, K. D., and A. Y. Nishimoto. Stimulation of Na’Ca” exchange in cardiac sarcolemmal vesicles by proteinase pretreatment. Am. J. Physiol. 243 (Cell Physiol. 12): C191-C195,1982. 25. Philipson, K. D., and R. Ward. Modulation of Na’-Ca”’ exchange and Ca” permeability in cardiac sarcolemmal vesicles by doxylstearic acids. Biochim. Biophys. Acta 897: 152-158, 1987. 26. Reeves, J. P., and K. D. Philipson. Sodium-calcium exchange activity in plasma membrane vesicles. In: Sodium-Calcium fixchange, edited by T. J. A. Allen, D. Noble, and H. Reuter. Oxford, UK: Oxford Univ. Press, 1989, p. 27-53. 27. Ruscak, M., J. Orlicky, M. Juhaszova, and J. Zachar. Na’Ca” exchange in plasma membranes of crayfish striated muscle. Gen. Physiol. Biophys. 6: 469-478, 1987. 28. Santer, R. M. Morphology and innervation of the fish heart. Adu. Anat. Embryol. Cell. Biol. 89: l-102, 1985.
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29. Tibbits, G. F., L. Hove-Madsen, and D. M. Bers. Ca” transport and the regulation of cardiac contractility in teleosts: a comparison with higher vertebrates. Can. J. ZooZ. 69: 2014-2019, 1991. 30. Tibbits, G. F., H. Kashihara, M. J. Thomas, J. E. Keen, and A. P. Farrell. Ca”’ transport in myocardial sarcolemma from rainbow trout. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R453-R460, 1990. Sl. Vemuri, R., M. E. Haberland, D. Fong, and K. D. Philipson.
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Identification of the cardiac sarcolemmal Na’-Ca” exchanger using monoclonal antibodies. J. Membr. BioL. 118: 279-293, 1990. 32. Vemuri, R., and K. D. Philipson. Phospholipid composition modulates the Na’-Ca” exchange activity of cardiac sarcolemma in reconstituted vesicles. Biochim. Biophys. Acta 937: 258-268, 1988. 33. Wang, C. S., and R. L. Smith. Lowry determination of protein in the presence of Triton X-100. Anal. Biochem. 63: 414-417,1975.
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of myocardial
Na+-Ca2+ exchange
GLEN F. TIBBITS, KENNETH D. PHILIPSON, AND HARUYO KASHIHARA Cardiac Membrane Research Laboratory, Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A 1 S6, Canada; and Cardiovascular Research Laboratory, University of California, Los Angeles, School of Medicine, Los Angeles, California 90024
Tibbits, Glen F., Kenneth D. Philipson, and Haruyo Kashihara. Characterization of myocardial Na’-Ca” exchange in rainbow trout. Am. J. Physiol. 262 (Cell Physiol. 31): C4ll-C417, 1992.-This study compared Na’-Ca”’ exchange from the hearts of rainbow trout with that from canines. In several respects, trout cardiac Na’-Ca2’ exchange is functionally similar to that from dogs and other mammals. Trout cardiac Na’-Ca” exchange is stimulated -200% after 30-min incubation with 10 pg/ml chymotrypsin at 21°C, similar to mammals. On the other hand, both the temperature and pH dependencies are strikingly different between the trout and canine myocardial Na+-Ca”+ exchange. While canine heart Na’-Ca”’ exchange exhibits a Q10 of >2 (similar to values observed in other mammals), that from trout is relatively insensitive to temperature with a Q10 of -1.2. The absolute rates of Na’-Ca” exchange in trout heart are four- to sixfold higher than that in mammals when measured at 7°C. Furthermore, the temperature insensitivity of trout myocardial Na’Ca” exchange is retained when the exchanger is reconstituted into an asolectin bilayer, suggesting that this property is intrinsic to the protein and not dependent on species differences in lipid bilayer composition. Trout Na+-Ca2+ exchange is not markedly stimulated by alkaline pH, in contrast to mammals, and this characteristic is also maintained after reconstitution. Western blots of trout cardiac sarcolemma run on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis react with antibodies raised against the canine Na+-Ca2+ exchanger with a similar pattern of bands (70,120, and 160 kDa). Furthermore, a cDNA probe from canine Na’-Ca” exchanger hybridizes on Northern blots of trout heart mRNA to a 7-kb band, similar to that in mammals. Thus, while important functional differences in Na’-Ca” exchange exist between trout and mammalian hearts, the molecular basis is not yet known. proteinase; ence
messenger
ribonucleic
acid; temperature
depend-
myocardial contraction is dependent on a transsarcolemmal influx of calcium (17), which has been proposed by Fabiato (13) to trigger the release of a greater quantity of calcium from the sarcoplasmic reticulum (SR). Na+-Ca2+ exchange is the primary mechanism of Ca”+ efflux across the sarcolemma (SL) in the mammalian heart and contributes, therefore, to mechanical relaxation (4, 7). Furthermore, it has been suggested recently that the Na+-Ca”+ exchanger contributes to SR Ca’+ release (18). Thus it is well established in mammals that Na’-Ca2’ exchange is an integral component in the regulation of myocardial contractility (26). Much less is known about Na’-Ca2’ exchange and contractility in the hearts of lower vertebrates. Electron microscopy of a number of poikilotherm hearts, including the trout, demonstrates both a sparsity of SR (28) in comparison to the mammalian heart and an absence of IN MAMMALS,
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transverse tubules. Furthermore, contraction in the amphibian (3) and teleost (12) ventricle appears to be relatively insensitive to ryanodine in doses known to block SR Ca2+release. Therefore, unless mitochondria contribute to myofilament Ca2+, an intracellular source of Ca2+ is of less significance in cardiac contraction in lower vertebrates. It should be noted, however, that in mammals a role for mitochondrial Ca2’ release in regulating contraction on a beat-to-beat basis has been dismissed based on a variety of arguments (8). Given these findings, it has been suggested that the magnitude of transsarcolemma1 Ca2+ transport in lower vertebrates is sufficient to support contraction (16, 30) and that Na’-Ca2’ exchange is the primary means of reducing cytosolic [Ca”‘] with each beat (29). Furthermore, active teleosts, such as rainbow trout and other Salmonidae, maintain reasonable cardiac outputs at temperatures (48°C) that are cardioplegic to mammals, demonstrating that Ca2’ transport systems are not dysfunctional in these species at low temperatures. We predicted, therefore, that trout Na+-Ca2+ exchange is comparatively more active than the mammalian exchanger under hypothermic conditions. As a consequence, this study sought to characterize the cardiac Na+-Ca2+ exchanger in SL from rainbow trout and compare it with that from mammals. METHODS Sarcolemmal isolation. Rainbow trout (Oncorhynchus mykiss) weighing 350-500 g were obtained from West Creek Trout Farm (Aldergrove, Canada) and maintained locally for a minimum of 2.5 wk at a temperature of +‘C. After the fish were killed with a sharp blow to the head, a ventral incision allowed the heart to be excised quickly. Typically, 12 trout ventricles were pooled, and each SL was isolated as described previously (30). SLs were isolated from the ventricles of mongrel dogs as detailed by Philipson and Ward (25). SL vesicles from both species were resuspended in loading medium (LM) which contained 140 mM NaCl and 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.4 at 21°C). Protein was determined in native sarcolemmal vesicles (NV) either by the Bradford (6) or Lowry (19) assays. Reconstitution. NV from both trout and canine hearts were reconstituted into asolectin membranes with modifications of a technique described previously (32). In brief, 33 ~1 of native SL (-3 mg/ml) were solubilized in 1 vol of 15 mM decylmaltoside and 140 mM NaCl. After spinning for 10 min at 178,000 g at room temperature in an Airfuge to remove nonsolubilized material, the supernatant was diluted with an equal volume of 140 mM NaCl and one-fourth volume of an asolectin mixture. The latter was produced by dissolving asolectin (50 mg/ml) in 7.5% Triton X-100, 100 mM MOPS-tris(hydroxymethyl)aminomethane (Tris) (pH 7.4 at 37”C), and 2.5 M NaCl and
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then sonicated for clarity. The resultant suspension was agitated in the presence of approximately one-fourth volume of BioBeads SM-2 for 20 min, and this step was repeated once to ensure removal of the detergents. After this treatment, the vesicles were diluted to 400 ~1 with LM and spun for 40 min in an Airfuge. The resultant pellet was carefully resuspended in 50 ~1 of LM. Na+-Ca” enchange. Sodium-dependent calcium uptake into NV was performed as described (24). In brief, 5 ,ul of Na’loaded sarcolemmal vesicles were suspended on the wall of a polystyrene tube containing 245 ~1 of uptake medium maintained at 21°C (unless indicated otherwise). The medium contained either 140 mM KC1 or NaCl, 2 &i 45Ca2+, 0.5 PM valinomycin, 20 PM 40Ca”‘, and, unless indicated otherwise, 10 mM MOPS (pH 7.0 at 21°C). In experiments in which the temperature dependence was to be determined, the vesicles were maintained at 4°C until suspended above the reaction media maintained at the desired temperature. In assays in which the extracellular pH (pH,) dependence was to be evaluated, 10 mM Tris maleate was used in lieu of MOPS and the pH was varied from 5.0 to 9.0. The uptake was initiated by vortex mixing and quenched at 2 s by the addition of 30 ~1 of a solution containing 140 mM KC1 and 1 mM LaC&. A 2209~1 aliquot was filtered (0.45 pm Millipore), and the filters were washed with 3 ml of a rinsing solution (containing 140 mM KC1 and 1 mM LaClJ twice. The filters were dried and then counted. The uptake by vesicles diluted into NaCl represented blanks and was subtracted for all data points. This allows for the correction of 4FiCa’2+ that was bound superficially to the sarcolemma or had permeated the vesicle by some pathway other than Na’-Ca”’ exchange. In all cases, this value was no greater than 10% of that obtained with K’ dilution. The Na+-dependent Ca”’ uptake into reconstituted vesicles (RV) was performed in a similar fashion as that in native vesicles except that 1) 3 ~1 of RV were used, 2) the reaction was quenched with 140 mM KC1 and 10 mM ethylene glycolbis(P-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 3) the filters had a pore size of 0.22 pm, and 4) the filters were rinsed with 140 mM KC1 and 1 mM EGTA maintained at 4°C. Protein was determined in RV by the method of Wang and Smith (33). Chymotrypsin treatment. NV from both trout and canine hearts were incubated with 10 pg/ml chymotrypsin for 30 min at 21°C. After this period of time, an aliquot of SL was mixed with the sample buffer in preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described, and the remainder was immediately used for Na’dependent Ca” uptake experiments. The latter was determined at 21°C and pH, 7.0. SDS-PAGE and Western blots. SDS-PAGE was performed on NV from trout and canine hearts as described (23) in 7.5% polyacrylamide. The proteins were transferred to nitrocellulose membranes electrophoretically and reacted with antibodies raised against the purified canine exchanger (23). Isolation of mRNA and Northern blots. Isolation of mRNA from both trout and canine heart followed the procedure of Chomczynski and Sacchi (10). Briefly, trout hearts were excised and frozen immediately in ethanol chilled with dry ice and then maintained at -70°C. About 2 g of frozen hearts were added to 30 ml of a denaturing solution (4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol) in a Waring blender and immediately homogenized for two bursts of 30 s each. The final pellet was dissolved in 0.5 ml diethyl pyrocarbonate (DEPC)-treated water. A l-p1 aliquot of the total RNA was analyzed spectrophotometrically and the remainder was used to isolate poly(A)+ RNA by oligo(dT) chromatography as described by Aviv and Leder (1). A l-p1 aliquot of the poly(A)+ RNA was also analyzed spectrophoto-
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metrically and then stored in liquid N, until used. RNA was denatured by heating for 5 min at 65°C in a solution containing formaldehyde and formamide. About 5 pug of denatured RNA were added to each well of a 1% agarose, 2.2 M formaldehyde gel. After separation, the RNA was transferred passively to a nitrocellulose membrane. A ‘31”C after reconstitution compared with the native membrane. The pH dependence of the Na’-Ca” exchanger from both species in native sarcolemmal vesicles is shown in Fig. 4. In the canine heart, there is an approximate 270% stimulation of Na’-Ca2’ exchange by increasing the pH from 7.0 to 9.0. No stimulation was observed in the trout heart Na’-Ca2’ exchange. A qualitatively similar result was obtained with the Na’-Ca2+ exchangers from both species after being reconstituted into asolectin vesicles (Fig. 5). Chymotrypsin, at a concentration of 10 pg/ml, stimulated the trout and canine Na+-dependent Ca”+ uptake in NV by -194 and 200%, respectively, as shown in Fig. 6. In both species, it was noted that this stimulation was associated with a more intense 70-kDa band and reduced intensity of the 120-kDa band in Western blots (data not shown). A Western blot using antibodies raised against the purified canine Na+-Ca2+ exchanger and SL from trout and canine hearts is shown in Fig. 7. The pattern of banding was similar in both species, with bands consistently appearing at 70, 120, and 160 kDa. The total RNA yield was 0.88 and 2.25 pg/mg wet wt in the canine and trout hearts, respectively. The 260/280
Trout Canin
I
1
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35
Temp
(lOOO/‘K)
Fig. 2. Arrhenius plot of the data shown in Fig. 1. Na+-dependent Ca2+ uptake was normalized for the rate at 37°C. Derived Qlo values of Na’Ca”’ exchange were 1.2 in the trout (7-37°C) and 2.0 (Zl-37°C) and 5.5 (7-21°C) in the canine heart. NV, native sarcolemmal vesicles.
c413
TROUT
(OC)
Fig. 3. Initial rates of Na+-dependent Ca”’ uptake in sarcolemmal vesicles (RV) as a function of temperature, for the rate at 21°C. [Ca’+], was 20 PM, n = 4 different for each group, and the reaction time was 2 s. RV were described in METHODS and maintained at 4°C until the initiated. 100% equals 8.4 and 8.6 nmol. mg protein-’ l s-l canine RV, respectively.
reconstituted normalized preparations prepared as reaction was in trout and
ratio was 1.60 and 1.74 for canine and trout RNA, respectively. In the agarose gels of poly(A)+ RNA, the 28s ribosomal RNA from the trout heart (-4.0 kb) always migrated farther than that from the canine heart (-4.4 kb). The canine heart 28s rRNA in all cases migrated the same distance as that from rat heart, brain, and adrenals, as well as rabbit heart and dog pancreas (data not shown). In the Northern blot, the cDNA probe from the canine heart Na’-Ca2’ exchanger hybridized nonspecifically to the 28s rRNA from both trout and canine heart. Although faint in the autoradiograph of the Northern blot shown in Fig. 8, the cDNA probe from canine heart Na+-Ca”+ exchanger routinely hybridized to a -7-kb band of trout mRNA, similar to mammals. DISCUSSION
The temperature dependence of Na+-Ca2+ exchange in the canine heart was similar to that observed by others using a variety of mammals, including dogs (5). Bersohn et al. (5) recently demonstrated that the amphibian heart Na+-Ca2+ exchange was less temperature dependent than that in mammals. The Arrhenius plot for Na+-Ca2+ exchange showed clear breaks at -22°C for both mammalian and amphibian hearts. In the present study, the teleost cardiac Na’-Ca”’ exchange shows even less temperature dependence than that of the frog and no obvious breakpoint in the Arrhenius plot. These properties were maintained in asolectin vesicles in all species. Therefore,
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0 l
Trout Canine
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NV NV
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0
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50 -CT 0
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Fig. 6. Initial rates of Na+-dependent Ca2+ uptake in NV before (-CT) and after (+CT) chymotrypsin treatment as described in METHODS. [Ca2+10 was 20 PM, n = 3 different preparations for each group; temperature was 21”C, and the reaction time was 2 s.
Fig. 4. Initial rates of Na+-dependent Ca*+ uptake in NV as a function of pH, normalized for the rate at pH 7.0. 100% equals 3.1 and 3.4 nmol. mg protein-‘.s-’ in trout and canine NV, respectively. [Ca2+10 was 20 PM, n = 4 different preparations for each group; temperature was 21”C, and the reaction time was 2 s.
-160 -120 200 0 c z a g 150
l
Trout Canine
RV RV
- 70
be
Tr
~,
,
0 5
7
Ca
Fig. 7. Western blot of native sarcolemma from trout (Ti; 23.4 pg) and canine (Ca: 9.7 ue) hearts run on 7.5% SDS-PAGE. Primarv I antibodies w&e raised & rabbits against purified canine cardiac Na+Ca” exchanger as described (23). Second antibody was goat anti-rabbit conjugated to horseradish peroxidase. Numbers on right refer to relative molecular mass (in kDa).
9
PH Fig. 5. Initial rates of Na+-dependent Ca*+ uptake in RV as a function of pH, normalized for the rate at pH 7.0. [Ca2+10 was 20 PM, n = 4 different preparations for each group; temperature was 21”C, and the reaction time was 2 s.
the species differences in temperature dependencies of Na+-Ca2+ exchange are apparently independent of possible differences in bilayer composition and must reflect differences in protein structure. In both Salmonidae (20) and mammals (5), the Michaelis constant for Ca2+
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MYOCARDIAL
NA+-CA’+
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4.4
Fig. 8. Autoradiograph of Northern blots of agarose gels of 5 pg of mRNA isolated from total RNA by oligo(dT) affinity chromatography from trout (Tr) and canine (Ca) hearts. The 32P-labeled probe was a 200-bp segment of clone A4 described by Nicoll et al. (21) from cDNA of the canine heart Na+-Ca2+ exchanger. Hybridization was performed as described in METHODS. Numbers on right refer to size of mRNA (in kb), and probe hybridizes to -7-kb mRNA segment in both trout and canine hearts.
[K,(Ca)] of Na+-Ca2+ exchange is unaffected by temperature, and thus the observed effects of temperature are due strictly to changes in maximum velocity (If,,,). The species variation in temperature dependence is an important physiological difference because cold-adapted Salmonidae maintain relatively high cardiac outputs at temperatures as low as 4°C. If Na+-Ca2+ exchange is responsible for the majority of cytosolic Ca2+ removal with each beat, as has been suggested (30), then this represents an important evolutionary adaptation. Na+Ca2+ exchange from crayfish skeletal muscle (27) has a reasonably similar temperature profile as that for trout myocardial Na+-Ca2’ exchange. In both trout cardiac (20) and crayfish skeletal muscle Na+-Ca2+ exchange (27), there was an obvious decline in activity when the membranes were maintained at >25”C for more than a brief period. Thus, in the present study, the temperature profile was determined with an exposure to the reaction temperature of no more than 2 s, and also the chymotrypsin proteolysis was carried out at 21°C. While a teleological explanation for the differences between poikilo- and homeotherms is apparent, a mechanistic interpretation is not. On the basis of the temperature profiles of cardiac Na+-Ca2+ exchange activity, there appears to be at least three different classes (mammalian, amphibian, and teleost) of exchangers that presumably reflect differences in the primary structure of the protein. Similar temperature-dependent species differences have been observed for the Ca2+ sensitivity of the cardiac contractile element (14). It should be noted, however, that trout p-nitrophenyl phosphatase (K+-pNPPase), a sarcolemmal protein, exhibits a temperature dependence radically different from that of the Na+-Ca2+ exchanger and not unlike that of the mammalian exchanger (G. F. Tibbits, unpublished observations). The lack of stimulation of trout cardiac Na+-Ca2+ exchange by alkaline pH was unexpected. The stimula-
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tion of canine cardiac Naf-Ca2+ exchange by alkaline pH observed in this study is similar to that observed in previous studies (22) on mammalian hearts. Similarly, this stimulation is observed when the exchanger is reconstituted into asolectin (32), indicating that this property is intrinsic to the protein itself. The alkaline-induced increase in Na+-Ca2+ exchange has also been observed in other mammalian tissues (26) as well as in invertebrates including squid giant axons (2, 11) and crayfish skeletal muscle (27). The stimulation of the cardiac exchanger is the result of lowering the &(Ca) of the exchanger at alkaline pH (22). It was postulated (22) that a histidine residue near a Ca2+ binding site on the exchanger becomes deprotonated at higher pH and changes the conformation of the Ca*+ binding site. However, other possible hypotheses remain viable and therefore a mechanistic explanation of the species difference is not possible. A teleological explanation for the lack of stimulation of trout Na+-Ca2+ exchange by high pH is not apparent. It should be noted, however, that in poikilotherms in which body temperatures vary with ambient conditions, both pH, and intracellular pH also fluctuate in a predictable manner, -O.O17/“C rise in temperature (15). This relationship is known to covary with the temperature dependencies of the pK of imidazole and the neutral pH of water (15). Whereas the “normal” pH, at 37°C is 7.4, it is likely to be 7.8 at 4°C. In the present study, pH, was maintained at 7.0 while temperature was allowed to vary from 7 to 37°C to isolate the independent effects of pH and temperature. The differences in the temperature and pH dependencies of Na+-Ca2+ exchange between the trout and dog hearts cannot be attributed to differences in the sarcolemma1 isolation procedures. Properties of the canine Na+-Ca2+ exchange described in this study are qualitatively similar to those from a variety of mammalian species using different SL isolation procedures (26). Furthermore, an SL isolation procedure for rat hearts that was identical to that used for trout in the present study yielded qualitatively similar results to that from the canine heart (G. F. Tibbits, unpublished observations). Similarly, it is unlikely that the species differences in pH and temperature dependencies of Na+-Ca2+ uptake observed in the reconstituted vesicles are due to contaminating endogenous phospholipids. This is based on the facts that 1) the ratio of exogenous to endogenous phospholipid is in excess of 12:l and 2) exchange activity in the reconstituted vesicles has an absolute requirement for specific exogenous lipids (32). From studies of this type, there is no evidence that the endogenous lipids form an annulus around the protein that is difficult to replace with exogenous lipids during reconstitution. As shown in Fig. 6, the stimulation of trout myocardial Na+-Ca2+ exchange by limited chymotrypsin proteolysis is similar to that observed in mammals in both the present study and others (24). Both the higher and lower vertebrates can be distinguished from the Na+-Ca2+ exchanger of the invertebrate Artemia which is insensitive to limited proteolysis (9). A Western blot of both canine and trout SL run on SDS-PAGE and reacted with polyclonal antibodies raised in rabbits to Na+-Ca2+ exchanger purified from
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dog heart is shown in Fig. 7. The specificity of the polyclonal antibodies for the Na+-Ca”+ exchange protein has been demonstrated with two different monoclonal antibodies (Ref. 31, and Frank et al., unpublished observations). Because this reaction was not as pronounced in trout, as compared with dog, typically 2.5 times as much protein was used per lane for trout. This is likely due to a lower antigenicity of trout Na+-Ca2+ exchanger compared with dog but is also possibly due to lower exchanger density in the SL preparations. The K+-pNPPase activhigher compared with ity in canine SL was -25fold trout SL and the amount of purification was also comparably higher. The pattern of banding on the Western blot was similar in both species. Recently, the canine Na’-Ca’+ exchange cDNA was cloned and sequenced, and the deduced molecular mass is 108 kDa (21). The 120-kDa band may be the consequence of posttranslational modification such as glycosylation. Regardless, the native molecular weight of the Na+-Ca*’ exchanger appears to be similar in the two species. In agarose gels of poly(A)+ RNA, the 28s rRNA observable at 4.4 kb in mammals always migrated further in trout for reasons that are unknown. Northern blots of these gels are shown in Fig. 8. A cDNA probe from the canine cardiac Na+-Ca*+ exchanger hybridizes to trout mRNA with a band at -7 kb similar to that in mammals. This indicates that there is some homology between Na+Ca’)+ exchanger cDNAs from the two species, and the transcripts are approximately the same size. These experiments, in general, show a high degree of similarity in the properties of the cardiac Na’-Ca*’ exchangers from mammals and teleosts. These similarities include K,(Ca) (29), stimulation by both valinomycin (29) and limited chymotrypsin proteolysis, apparent molecular weight of the protein, and size of transcript. The most striking difference between the two species is the temperature dependence that is likely due to differences in the protein amino acid sequence. Because the mammalian Na+-Ca”+ exchanger antibodies cross-react with trout Na+-Ca”+ exchanger and because mammalian Na+Ca’+ exchanger cDNA hybridizes to trout Na’-Ca*’ exchanger mRNA, there is significant homology between the two species. A mechanistic interpretation of the different temperature profiles will require a more detailed knowledge of the structure of the myocardial Na+-Ca”+ exchangers. We gratefully acknowledge the generous support of the National Sciences and Engineering Research Council (Canada), the British Columbia Health Research Foundation (to G. F. Tibbits), and the National Institutes of Health (to K. D. Philipson). The contributions of Drs. Zhaoping Li, John Keen, Deb Nicoll, and Marion Thomas are appreciated. Address for reprint requests: G. F. Tibbits, Cardiac Membrane Research Laboratory, Kinesiology, Simon Fraser University, Burnaby, British Columbia V5A lS6, Canada. Received
20 June
1991; accepted
in final
form
28 August
1991.
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29. Tibbits, G. F., L. Hove-Madsen, and D. M. Bers. Ca” transport and the regulation of cardiac contractility in teleosts: a comparison with higher vertebrates. Can. J. ZooZ. 69: 2014-2019, 1991. 30. Tibbits, G. F., H. Kashihara, M. J. Thomas, J. E. Keen, and A. P. Farrell. Ca”’ transport in myocardial sarcolemma from rainbow trout. Am. J. Physiol. 259 (Regulatory Integrative Comp. Physiol. 28): R453-R460, 1990. Sl. Vemuri, R., M. E. Haberland, D. Fong, and K. D. Philipson.
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Identification of the cardiac sarcolemmal Na’-Ca” exchanger using monoclonal antibodies. J. Membr. BioL. 118: 279-293, 1990. 32. Vemuri, R., and K. D. Philipson. Phospholipid composition modulates the Na’-Ca” exchange activity of cardiac sarcolemma in reconstituted vesicles. Biochim. Biophys. Acta 937: 258-268, 1988. 33. Wang, C. S., and R. L. Smith. Lowry determination of protein in the presence of Triton X-100. Anal. Biochem. 63: 414-417,1975.
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