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Calcium-linked changes in myocardial metabolism in the isolated perfused rat heart1 N. S. DHALLA,". C . YATES,AND V. PROVEDA Pathophy,sic~lo,vvLahoratorg~,Department of Physiology, F ~ c u l t yof Medicine, University of Manitohn, Winnipeg, Man., Canada R3E OW3 Received August 18. 1976
DIIAILA, N . S., YAI'LS,J . C.. and PROVEUA, V. 1977. Calcium-linked changes in myocardial metabolism in the isolated perfused rat heart. Can. J. Physiol. Pharniacol. 55, 925-933. Rat hearts were perfused for 40 min with aerobic medium containing different concen(0-5 m M j and their abilities to take up and oxidize glucose, and to trations of c a l c i ~ ~ m produce lactate and glycerol were examined in addition to measuring glycogen, lipids. cyclic AMP, and high energy phosphate stores. 'Increasing the concentration of calcium was found to decrease myocardial glycogen but increase glucose uptake, glucose oxidation, and lactate release. A decrease in myocardial triglycerides and an increase in free fatty acid contents as well as glycerol release without any changes in cholesterol and phospholipid contents were observed upon increasing the concentration of external calcium. In comparison with the hearts perfused with Ca2+-free medium, the levels of creatine phosphate and ATP were lower and that of ADP higher in hearts perfused with medium containing 5 mM calcium. N o differences in AMP and cyclic AMP contents were seen among hearts perfused with different concentrations of calcium. The contractile activity initially increased upon increasing the concentration of calcium from 1.25 to 5 m M and then declined towards the control level. The hearts were unable to generate contractile force in the absence of calcium. whereas the contractile force decreased and then began to recover upon perfusing the hearts with 0.31 mM calciuin. These results indicate that elevated levels of intracellular calcium stimulate glycogenolytic, glycolytic, and lipolytic processes in myocardium directly. DIIAII it, N. S., YAIES,J . C. et P ~ o v aDA, V. 1977. Calcium-linhed changes in n ~ y o cardial metabolism in the isolated perfused rat heart. Can. J . Physiol. Pharmacol. 55, 925-933. Des coeurs de rat sont perfusks pendant 40 min avec un milieu akrobique contenant diverses concentrations de calcium (0.5 m M ) ; leurs capacitks de captation et d'oxydation du glucose, des lipides, de 1'AMP cyclique et des rCserves de phosphates B haute energie sont examinkes. L'augmentation de la colicentration en calcium diminue le glycogbne myocardique. mais augmente la captation de glucose, l'oxydation du glucose et la liberation de lactate. Une diminution des triglyc6rides myocardiques, une augmentation du contenu en acides gras libres et de la liberation de glycerol, sans changements du contenu en cholest61-01 et en phospholipides, sont observtes apres augmentation de la concentration extracellulaire de calcium. Par rapport aux coeurs perfus& avec un milieu ctCpourvu de calcium, les taux de cl-Catine phosphate et d9ATI' sont moins ilevts alors que ceux d'ABP sont plus klevks seulement dans les coeurs perfusis avec un milieu contenant 5 mM de calcium. Aucune difference de contenu cn AMP et en A M P cyclique n'est relevee dans les coeurs perfuse.; h concentration diffkrente de calcium. L'activiti contractile augmente initialernent apr6s augmentation de la concentration de calcium de 1.25 2 5 m M et diminue ensuite jusqu'aux valeurs temoins. Les coeurs sont incapables de developper une force contractile en l'absence de calcium, alors que la force contractile diminue, puis revient A la normale sous l'effet d'une perfusion du coeur avec 0.31 mM de calcium. Ces rksultats indiquent que des valeurs ClevCes du calcium intracellulaire stimulent les processus glycogCnolytiques, glycolytiques et lipolytiques dans le mymarde. [Traduit par le journal] ABBREVIATION: CrP. creatine phosphate. 'This work was supported by a grant from the Manitoba Heart Foundation. 'Please address correspondence to: Dr. Naranjan S. Dhalla, Professor of Physiology, Fdculty of Medicine, University of Manitoba, Winnipeg, Man., Canada K3E OW3.
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Calcium is considered to play a crucial role in the regulation of celIular function and metabolism (Basmussen et a&. 1972). A great deal of effort has been made for understanding the participation of calcium ions in a wide variety of events intinaately associated with contractile activity of myocardium, and it is now established that calcium links the processes of excitation and contraction (Langer 1968; Sonnenblick and Stana I969 ;Katz 1978; Reuter 1974). However, relatively little is known about the involvement of calcium in processes regulating myocardial metabolism. Since calcium is known to increase the activity of myocardial lipase (EC 3.1.1.3) and phosphorylase kinase (EC 2.7.6.38) phosphorylase systems (Neely, Roveto et a&. 1972; Jesnnok et ak. 1976; Friesen et al. 1969; Gross and Mayer 1975), it is conceivable that this cation may participate in the processes of Iipolysis and glycogenolysis in myocardiuln. To gain some information in this regard the effects of calcium on the abilities of the isolated heart to use endogenous lipids and glycogen, and to prodaacc glycerol and lactate were tested. The abilities of tlae hearts to take up and oxidize glucose were also examined in the presence of different concentrations of calcium in the perfusion medium. The myocardial contents of cyclic AMP and adenine nucleotides were measured because changes in their concentrations are known to have profound effects on the glycolytic, glycogenolytic, and lipolytic activities of myocardium (Haugaard and Hess 1965; Hiraams-Hagen 1967; Wobison et al. 1968; Neely, Roveto et al. 8972). The contractile activity of these hearts was monitored during the course of perfusion with medium containing different concentrations of calcium.
Male hooded rats weighing 300-350 g were decapitated. the hearts quickly removed and arranged for coronary perfrision by the Langendorff technique as described earlier (DhalIa ef al. 1973). The KrebsHenseleit solution, pH 7.4, containing 8 m M glucose and different eonccntrations of calcium was gassed with a mixture of 95% O2and 5% C02, and maintained at 37°C. The hearts were punctmed with the sharp end of a pair of small scissors to avoid accumulation of fluid in the ventricles. A resting tension of 1 g was applied to the heart and the contractile force was
monitored on a Grass polygraph via a force displacement transducer QFT.03).All hearts were equilibrated with the perftisisn medium containing 1.25 mM calcium POP 15 min in an open system. At the end of this period the hearts were switched to a closed perfusion system (Bleehen and Fisher 1954) containing 50 ml of the desired medium. The coronary Wow was maintained at 10 ml/min with the help of a peristaltic pump. Perfusate (0.5-1 ml) was withdrawn for biochemical analysis at different intervals of the perfusion; this fluid was not replaced but appropriate corrections were made while calculating the data. At the end of each experiment, the hearts were frozen for biochemical detcrrninaltion with a bvollenberger clamp precooled in liquid N,. Dry weight of a portion of the ventricle was obtained by keeping the tissue in an oven at 100°C for 2 days. Glycogen from myocardium was extracted with hot 30% KOM and measured by the glucose oxidase (EC' 1.1.3.4) method (Huggett and Nixon 1957). Glucose uptake and lactate release from the isolated rat heart were measured by the procedures described by Williamson ( 1966). "CO, production was measured according to the method described by Kreisberg and Williamson (1964) upon perfusing the hearts with a medium containing 8 m M [kT-l'C]gTucsse (0.15 p C i / n ~ l( I Ci = 37 GBq)). Phospholipid and neutral lipids were extracted and separated by the method of Folch et al. ( 1 957) and Bsrgstrom ( 1952). The phospholipid phosphonns, triglyceride glycerol, cholesterol, and free fatty acids were measured according to the procedures descrikcd elsewhere (Wells and Dittmer 6965; Sperry iind Webb 1950; Rapport and AIonzo 1955). The ]high energy phosphate stores (CrP, ATP. ADP, and AMP) were determined by the flusrometric methods (Fcdelesova and Dhalla 1971 ). The extraction and measurement of cyclic AMP were carried out by the methods of Brown et ul. ( 197 1) and Cilman ( B970), respectively.
Jn one set of experiments, rat hearts were perfused for 40 min with medium containing 0, 0.31, 1.25, and 5 mM calcium; the concentxations of glucose or lactate in the perfusate and the production of d4CC02were measured at different intervals and myocardial glycogen contents were determined at the end of the perfusion period. The data shown in Figs. 1 and 2 indicate that the cardiac glucose uptake and lactate output were increased upon increasing the concentration of calcium in the perfusion medium. An increase in the oxidation of glucose was evident since an increase in 14@Oip production froin [U-14G]glucose was observed upon increasing the concentration of calcium in the perfusion medium (Fig. 3). Myocardial levels of glycogen declined markedly upon perfusing
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DI-IALLA ET AL.
TlME (min)
FIG.I . GIucose uptake by the isolated rat heart perfused with different concentrations of calcium for a period of 48 min. Each value is an average of five experiments. Vertical bars indicate 2 SEM.
FIG.2. Lactate output by the isolated rat heart perfused with different concentrations s f calcium far a period of 40 min. Each value is an average of six experiments. Vertical bars indicate 2 SEM.
the hearts with elevated levels of calcium (Fig. 4) The second set of experiments was performed to determine changes in lipid contents and the ability of heart to release glycercd upon perfusion with medium containing 0, 0.3 1, 1.25, and 5 mM concentrations of calcium. The data in Fig. 5 indicate that the level af triglycerides, but not that of cholesterol, in hearts perfused with medium containing 0.31-5 mM Ca2+was lower than that in the Ca2+-freeperfused hearts. On the other hand, thc concentration of free fatty acids, but not of phospholipids, was higher ( B < 0.05) in hearts perfused with 0.31-5 ntM ca?+in cornparison with that in hearts perfused with Ca"-free medium (Fig. 6 ) . The amounts of glycerol released from the hearts perf used with medium containing 0.31 -5 mM CaN were also higher than those from the hearts perfused with Ca2+-free medium (Fig. 7). Tn the third set of experiments, high energy phosphate stores and cyclic AMP concentrations in the hearts were measured after perfusion with different concentrations of calcium
for 40 min. The results in Table 1 reveal that CrP and ATP levels were lower and ABP levels higher in hearts perfused with 5 mM Ca2+ in comparison with hearts perfused with Ca2+-f ree medium. No change in AMP and cyclic AMP contents was seen in hearts upon perfusion with different concentrations of Caw. The time course of changes in contractile force due to perfusing the hearts with different concentrations of calcium is shown in Fig. 8. The hearts were unable to generate contractile forcc within 30 s to 1 min when perfused with Ca2+-free medium. It was interesting to note that the contractile force declined by about 75% within 2-4 min but thereafter started to recover towards the control levels over a period of 36 nlin when the hearts were perfused with 0.31 mM Ca9* Marked increments in the contractile force were seen upon perfusing the hearts with 2.5 or 5 mM Ca2+ within 2-4 rnin but thereafter started to decline towards the control leerel within $0 min of perfusion. A similar pattern of changes in the contractile force due to pelfusion with media containing
CAN. J. PHYSIOL. PHARMACOL. VOL. 55.
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TIME
(min)
F ~3. ~I .~ ~ production O , by isolated rat heart perfused with different concentrations of calcium for a period of 40 min. Each value is an averagc of four experiments. Vertical bars indicate 2 SEM.
1977
FIG.4. Glycogen levels in the isolated rat heart perfnsed with difTcrent concentrations of calcium for a period of 40 min. Each value is a mean & SE of I I
described here is a mean t SE of four to seven experiments. 'To determine if the observed effects sf calciu ~ narc mediated through the endogenous catecholamines - adrenergic receptor activation, ~ 0 1 1 1effects ~ of calcium were tested on hearts pcrfused with medium containing propranololl, a well-known P-adrenergic receptor blocking agent. It was found that perfusing hearts with 50-100 p M gropranolol, which completely blocked the positive inotrspic effect of 0.5 p g epinephrine, failed to alter the positive inotropic effect (68 t 3 % increase at 10 rnin), lactate production (26 1.7 pmol lactate/g dry heart weight per 10 rnin), and glycerol release (1.5 t 0.2 pmoI glycerol/g dry heart weight per 10 nmin) due to 5 mM Ca". The results described SE sf four experiments. here are mean
0-5 mM calcium was observed in hearts paced electrically at 250 beats/mimn at a voltage just above the threshold. Thus, it is unlikely that the observed changes in contractility are due to any alterations in heart rates. The time course of changes in myocardial metabolism due to 5 mM Ca" was also investigated. Switching the hearts to medium containing 5 mM Ca" was found to increase the contractile force by 79 t 0.9% of the control levels in 2 rnin without any significant changes in the coltcentrations of cyclic AMP, AMP, or ATP; however, the levels of ADP were increased by 21 -t- 0.5% of the control values. Significant reductions in the glycogen (25 31.3% decrease) and CrP (19 +- 1.5% decrease) contents were seen at 5 and 10 min after starting the perfusion with 5 mM CawC, Discussion respectively. whereas significant decreases in triglyceride (21 It 0.7% decrease) and ATP In this study we have shown that increasing (22 t 0.8% decrease) content were observed the concentration of calciunl in the perfusion after 20 rnin of pedusing the hearts with 5 mM medium resulted in decreasing triglyceride and ca2+ . It should be mentioned that each value glycogen contcnts of the heart. This lipolytic _+
IIHALLA ET AL.
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TRlGLYCERlDES
C
(pmol/o
k
CHOLESTEROL
dry
( p r n o ~ / ~d r y
FREE FATTY heart
vrrigkt )
h a a r t ueiph8 )
.I
I
.)
ACIDS
(.J J R R O I / Qd r y
( p r p p o ~ / ~d r y
heart
heart
weight
1
weigh?)
1 4
C ~ + C B N C L N T W A JfmM) IOM
FIG.5. Triglyceride m d cholesterol levels in the isolated rat heart perfused with different concermtrations of calcium for a period of 40 min. Each value is a mean zk SE of nine experiments.
effect of calciurn was also obvious from the elevated levels of free fatty acid without any changes in the phospholipid or cholesterol contents in myocardium as well as increased release of glycerol in the perfusion medium upon perfusing the hearts with increasing concentrations of calcium. Although the glycogenslytic effect of calcium was associated with an increased production of lactate by myocardium, glucose uptake was also increased upon elevating the concentrations of calcium in the perfusion medium. Raising the concentration of external calcium has been shown to enhance the permeability of glucose in skeletal muscle ( WolHoszy and Narahara 196'7 ) . The observed increase in glucose uptake may probably be due to the inlnibitory effect of calcium on the Na+-K+ ATPase, which has been considered to be intimately involved in regulating the transport of glucose across the cell membrane (Schwartz et al. f 972). On the other hand, the glycogenolytic and lipolytic effects of calcium are mediated due to the activation of phosphorylase and triglyceride lipase, respectively
FIG.6. Free fatty acid and phospholipid levels in the isolated rat heart perfused with different somentratisns of calcium for a period of 40 min. Each value is a mean 9 SE of nine cwperirnents.
(Friesen eir ah. 1969; Jesmok et a&. 1976). Decrease in glycogen and triglyceride contents, and increase in glucose uptake, g%ucose oxidation, glycerol release, arad lactate output by the isolated hearts have also been shown to occur in the presence of hormones such as epinephrine and glucagon (@orriblath et a!. 1963; \Vi8liamson 1964, 1966; Mrcisberg 1966; Kreisberg and Willian~strnf 964). These metabolic effects of hormones are elicited through the participation of cyclic AMP (Himms-Hagen 1967; Robison et ab. 19681. However, glycolytic, glycogenolytic, 2nd lipo'Lytic actions sf calcium cannot be considered to be mediated through cyclic AMP because its concentration did not change upon perfusing the hearts with elevated concentrations of calcium. Since perfusing the hearts with propranolol did not prevent the calcium-induced glycerol release or lactate production, It is unlikely that these metabolic effects of calcium are a consequence of norepinephrine release from the nerve endings present in the isolated heart preparations. Increasing the extracellular concentrat ion of calcium has been shoavw to increase myocardial
930
CAN. J. PEWSIBL. BHARMACOE. VOE. 55. 1977
TABLEI. High energy phosphate stores and cyclic AMP content of the isolated rat heart perfused for 40 min
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with mediun-i containing different concentrations of calcium Cowcn. of Catf in the perfusion medium, rnM
High energy phosphate cs~npoundsu,ymol/g dry heart weight CrP
AMP
ADP
ATP
Cyclic A M P prno8i.g dry heart weight
=Each value is a mean f- Sk; of sin experiments. bSisnifmcaratly different (P < 0.05) from the values obtniraed with Cal+-free (0 mM) medium.
P
GLYCEROL (ymol/g
RELEASED dry
heart
weight
1
FIG.7. Giycerol release from the isolated rat heart perfused with different concentrations of calcium for a period of 40 min. Each value is an average of nine experiments. Vertical bars indicate 2 SEM.
oxygen consumption (Sonnenblick et al. 1965) and this could be interpreted to reflect an increase in the processes involved in ATB production. This view is supported by our finding that an increase in l9CO2production from 1' 4Cjglucose was seen in the isolated hearts upon perfusion with elevated levels sf calcium. On the other hand, raising the external calcium is expected t~ increase rnyofibrillar ATBase activity
(Katz 1970) and result in an increased use of ATP. The rates of splitting of CrB and ATP in beating rabbit atria have been reported to increase upon increasing the concentration of calcium in the bathing medium (Schildberg and Fleckenstein 2 965 ) . The increased amounts of energy and ABP, which would be available due to increased hydrolysis of ATB, will be used for greater development of the contractile force
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DHAkLA ET A k .
0
8
16
24
32
QB
T l M E (rnin)
FIG.8. Contractile force of the isolated rat heart perfused with different concentrations of calcinm for a period of 40 min. Each value is an average of 25 experiments except that the values for 2.5 mM Ca2+are from four experiments. Vertical bars indicate 2 SEM.
and stimulating the mitochondria1 respiratory activity, respectively. Since increasing the concentration of calcium from 0 to 1.25 rmkl did not change the levels sf CrP or ATP in the perfused heart, it appears that ATP use due to elevated myofibrillar ATPase activity is balanced with ATP praduction due to an increased operation of rnitochondrial oxidative phosphorylation activity. It should be noted that both CrP and ATP levels declined in hearts perfused with medium containing 5 mM calcium. Such a change is most likely due to an insufficient process of ATB production, because under this condition myocardium is loaded with calcium (Tomlinson et wl. 1974) which may depress mitochondria1 oxidative ghosphorylation activity (Wrogernann and Pena 1976). At any rate, it is unlikely that the observed glycogenolytic, glycolytic, and lipslytic actions of calcium can be explained on the basis of changes in the concentrations of ATP, ABP, and AMP (Haugaard and Hess 1965; Neelly,
Benton et wl. 1972) since the levels of these nucleotides did not change upon perfusing the hearts with 0 to 1.25 rnkf calcium. A parallel relationship between external calcium and force of cardiac contraction has been clearly demonstrated (Loewi 1918; Luttgau and Niedergerke 6958). Such an increase in contractile tension by calcium has been suggested to be due to an increase in ATP use as well as conversion of chemical into mechanical energy (Furchgott and Lee I96 1) . However, in this study we have shown that the isolated hearts exhibit a remarkable ability of adaptation with respect to maintaining their contractile activity when perfused for prolonged periods with different concentrations sf calcium. For example, the contractile force decreased initially but recovered towards normal upon starting the perfusion with 0.31 mM calcium. Likewise, the contractile force increased initially but declined towards normal upon starting the perfusion with 2.5-5.0 mlM calci-
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CAN. J. PWSIOE. PHARMACOL.
urn. Whether these changes in mvocardiaI " contractility during the course sf perfusion with low or high concentrations sf calcium are due to some adjustments in the fluxes of calcium across different nsembranes or in the processes concerned witla energy production and use remain to be determined. It should be pointed out that increasing the extracellular concentration sf calcium has been shown to increase calcium influx (Winegrad and Shanes 1962) and this may elevate the intracellular level of calcium in the nsyocardial cell. Elevated levels sf the in-mtraicellularcalcium can also be assumed from the increased abilities of the hearts to generate higher contractile forcc tapon perfusion with high corpcentrations of extrace%lular caIcium. T~PUS, an increase irn the intracellular concentration of calciurn can be conceived to activate phosphorylase kinase directly and cause glycogenolysis (Gross and Mayer 1975). A similar kinapse syster-st has also been propsscd for the activation of the myocardial triglyceride lipase system by callcihan~(Sesmak et ab. 1974). The lipolytig: and g8ycogenolytic efFects of increased ventricular pressure (Crass et a/. 1969, 197l; Neely et a/, 1969; Neely, Denton sf aH. 19721, in which condition cab cium movements within the myocardiunn must be augmented, for increasi~sgcontractility can also be explained on the basis of calcium involvement. Since epinephrine is known to increase calcium influx (Remter 1974), some contribution of calcium in hormone-induced glycogenoIysis and Iipolysis in heart cannot be ruled out.
BHFEHEN, N . Me, and FISHER, R. B. 1954. The action of insulin in the isolated rat heart. J . Physiol. (London), 123, 260-274. B~MGSTROM, B. 1952. Investigation on lipid separation methods. Separation of phaspkolipids from neutral Pat and fatty acids. Acta Physiol. Scand. 21, 181-110. BROWN,B. L., ALBANO,J . D. M.,Earn~s,R. P., and SGIIEWZH. A. M. 1971. A simple and sensitive saturation assay method for the measurement of adenosine3':s'-cyclic naonophosphate. Biochem. J. 121, 561562. CORNWLATH, M., RANDLE, P. J., PARMEGGIANI, A., and MORGAN. H. E. 1963. ReguHa tion of glyccsgenolysis in rnuscIe: effect of giucagon and anoxia on lactate producta'sn. glycogen content and phosphorylase activity in the perfused rat heart. J . Biol. Chern. 233, 1592-1 597. CRASS,IPI, M. F., MCCASKELL, E. S., and Snirp~,J. C. 1969. Effects of pressure development on glucose
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and palmitate metabolism in perfused heart. Am. J. Physiol. 216, 1569-1 576. CRASS.111, M. F., M@CASKEI,E, E. S., SHIPP,%.C . , and MURTHY,$7. K. 1971. Metabolism of endogenous lipids in cardiac mtlscle: Effect of pressure development. Am. J. PhysioI. 228,428-435. DHAELA, N. S., MATOUSEIEK, R . F., SUN, C. N., and BESON,R. E. 1973. hfetabelic, ultrastructural and rnechanicaB changes in the isolated rat heart perfused with aerobic medium in the absence or presence of glucose. Can. J. PhysioI. Pharmacol. Sf. 590-603. FEDEIESOVA, M., and DIIALLA, N. S. 1971. High energy phosphate stores in the hearts of genetically dystrophic han-ssters. %.Mol. Cell. CarclioB. 3, 93-182. Fo~c-11, J., 1 ~ ~M., s and . SEOAN-STANILY, G. W. 1954. A simple method for the isolation and purification of total lipids from animal tissues. J . Niol. Chem. 226,497-589. FRICSEN,A. 3. D., OLIVER,W., and ALLEN,G . 1969. Activation of cardiac glycogen phosphorylase by calcium. Am. J. Physiol. 217, 445-450. FURCPI~OTT, R. F., and LEE,,K. S. 1961. Nigh energy phosphate and the force of contraction of cardiac muscle. Circulation, 24, 416-428. GII-MAN,A. G . 1940. A protein binding assay for adenosine 3"5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67, 305-3 12. Claoss, S. W., and MAYER,S. E. 1975. Phosphorylase kinase mediating the effects of cyclic AMP in rnrascle. Metabolism, 24, 369-380. HAUGAARD, N., and HESS, M. E. 1965. Actions of autonomic dmgs on phssphorylase activity and function. Pharrnaeol. Rev. 17, 27-70. HIMMS-MAGEN, 9. 8967. Sympathetic regulation of rnetabolisrn. Pharmacol. Rev. 19, 367-461. Moe~oszu.J. 0., and NARAHARA, £3. T. 1967. Enhanced perrneztbi8ity to sugar associated with muscle contraction. Studies sf the role s f Cab-. I . Cen. Physiol. 568. 55 1-562. HUGGET, A. St. G., and NIXON,D. A. 1957. Use of glucose oxidase, peroxidase and 0-dianisidine in determination of blood and urinary glucose. 1,ancet ii, 368-370. JESMOK, G. J . , MOGELNICKI, S. W., LECH,J. J., and CAIVERT, D. N. 1976. Effect of polyamines, ionc and epinephrine on triolein hydrolyzing activity of rat venbricallar extracts. J. Mol. Cell. Cardiol. 8, 283-298. KATZ,A. M. 1970. ConItractiIe proteins of the heart. Physiol. Rev. 50, 63-158. KREISBERG, R. A. 1966. Effect of epinephrine on n-syocardial triglyceride and free fatty acid utilization. Am. J, Physiol. 210, 385-389. KREISBERG, R. A.. and WILLIAMSON, J. R. 1964. Metabolic effects of glucagon in the perfused rat heart. Am. J. Physiol. 207, 721-727. LANGLR, G. A. 1968~Ion fluxes in cardiac excitation and contraction and their relation to myocardial contractility. Physiol. Rev. 48,708-757. I[,oE.wI, 0. 19 18. Uber den Zusammenhang Zwischen Digitalis und KaIziumwirkung. Arch. Exp. Patkol. Pharrnak. 882, 131-1-98.
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topics in membranes and transport. Vol. 3. Edited by LUTTC~AU, H. C., and NIEDERGERKE, R. 1958. The F. Bronner and A. Kleinzeller. Academic Press Inc., antagonism between Ca" and Na' ions on frog's New York. pp. 1-82. heart. J. Physiol. (London), 143, 486-505. NEELY,J. R., BOWMAN, R. H., and MORGAN, H. E. SONNENBLICK, E. H., ROSS, JR., J., COVELL.J. W., 1969. Effects of ventricular pressure development KAISER,G o A., and BRAUNWALD, E. 1965. Velocity and palmitate on glucose transport. Am. J. Physiol. of contraction as determinant of myocardial oxygen 216,804-81 1. consumption. Am. J. Physiol. 203,919-927. NEELY,J. R., DENTON,R. M., ENGLAND, P. J., and SONNENBI-ICK, E. H., and STAM, JR., A. C. 1969. RANDLE, P. J. 1972. The effects of increased heart Cardiac muscle: Activation and contraction. Annu. work on the tricarboxylate cycle and its interaction Kev. Physiol. 31, 647-674. with glycolysis in perfused rat heart. Biochem. J. SPERRY, W. A., and WEBB,M. 1950. A revision of the 128. 147-159. Schoenheimer-Sperry method for cholesterol deterNEELY,J. R., ROVETO,M. J., and ORAN,J. F. 1972. mination. 9. Biol. Chem. 187, 97-106. Myocardial utilization of carbohydrate and lipids. TOMLINSON, N. S. C. W., YATES,J. C., and DHALLA, Prog. Cardiovasc. Dis. 15, 389-399. 1974. Relationship among changes in intracellular RAPPORT,M. M., and ALONZO,N. 1955. Photometric calcitim stores, ultrastructure, and contractility of determination of fatty acid ester groups in phosphomyocardium. In Myocardial biology. Vol. 4. Edited lipids. J . Biol. Chem. 217, 193-198. b y N. S. Dhalla. University Park Press, Baltimore. RASMUSSEN, W., GOODMAN, D. B. P., and TENENHOUSE, pp. 331-345. A. 1972. The role of cyclic AMP and calcium in cell WELLS,M. A., and DITTMER,J. C. 1945. The quantitaactivation. Crit. Rev. Biochem. 1,95-148. tive extraction and analysis of brain polyphosphoREUTER,W. 1974. Exchange of calcium ions in the inositides. Biochemistry, 4,2459-2467. mammalian myocardium. Mechanisms and physioWILLIAMSON, J. R. 1954. bletabolic effect of epinephlogical significance. Circ. Res. 34, 599-605. ROBISON,G. A., BUTCHER,R. W., and SUTHLRLAND, rine in the isolated perfused rat heart. I. Dissociation of glycogenolytic from metabolic stimulatory E. W. 1968. Cyclic AMP. Annu. Rev. Biochem. 37, effect. J. Biol. Chem. 239, 2721-2729. 149-174. 1966. Metabolic effects qf epinephrine in the SCHILDBERG, F. W., and FLECKENSTEIN, A. 1965. Die perfused rat heart 11. Control steps of glucose and Bedentung der extracellularen Calciumkonzentraglycogen metabolism. Mol. Pharmacol. 2, 206-220. tion fur die Spaltung von energiereichem Posphate WINEGRAD, S., and SHANES,A. M. 1962. Calcium flux in ruhenden under tatigem Myokardgewebe. and contractility in guinea pig atria. J. Gen. Physisl. Pfluegers Arch. Gesamte Physiol. Menschen Tiere, 45,371-394. 283, 137-1 50. SCHWARTZ, A.. LINDENMAYER, G. E., and ALLEN,J. C. WROGEMANN, K., and PENA,S. D. 1976. Mitochondria1 calcium overload: A general mechanism for cell 1972. The Na+,K+-ATPase membrane transport necrosis in muscle diseases. Lancet i, 672474. system: Importance in cellular function. In Current
Calcium-linked changes in myocardial metabolism in the isolated perfused rat heart1 N. S. DHALLA,". C . YATES,AND V. PROVEDA Pathophy,sic~lo,vvLahoratorg~,Department of Physiology, F ~ c u l t yof Medicine, University of Manitohn, Winnipeg, Man., Canada R3E OW3 Received August 18. 1976
DIIAILA, N . S., YAI'LS,J . C.. and PROVEUA, V. 1977. Calcium-linked changes in myocardial metabolism in the isolated perfused rat heart. Can. J. Physiol. Pharniacol. 55, 925-933. Rat hearts were perfused for 40 min with aerobic medium containing different concen(0-5 m M j and their abilities to take up and oxidize glucose, and to trations of c a l c i ~ ~ m produce lactate and glycerol were examined in addition to measuring glycogen, lipids. cyclic AMP, and high energy phosphate stores. 'Increasing the concentration of calcium was found to decrease myocardial glycogen but increase glucose uptake, glucose oxidation, and lactate release. A decrease in myocardial triglycerides and an increase in free fatty acid contents as well as glycerol release without any changes in cholesterol and phospholipid contents were observed upon increasing the concentration of external calcium. In comparison with the hearts perfused with Ca2+-free medium, the levels of creatine phosphate and ATP were lower and that of ADP higher in hearts perfused with medium containing 5 mM calcium. N o differences in AMP and cyclic AMP contents were seen among hearts perfused with different concentrations of calcium. The contractile activity initially increased upon increasing the concentration of calcium from 1.25 to 5 m M and then declined towards the control level. The hearts were unable to generate contractile force in the absence of calcium. whereas the contractile force decreased and then began to recover upon perfusing the hearts with 0.31 mM calciuin. These results indicate that elevated levels of intracellular calcium stimulate glycogenolytic, glycolytic, and lipolytic processes in myocardium directly. DIIAII it, N. S., YAIES,J . C. et P ~ o v aDA, V. 1977. Calcium-linhed changes in n ~ y o cardial metabolism in the isolated perfused rat heart. Can. J . Physiol. Pharmacol. 55, 925-933. Des coeurs de rat sont perfusks pendant 40 min avec un milieu akrobique contenant diverses concentrations de calcium (0.5 m M ) ; leurs capacitks de captation et d'oxydation du glucose, des lipides, de 1'AMP cyclique et des rCserves de phosphates B haute energie sont examinkes. L'augmentation de la colicentration en calcium diminue le glycogbne myocardique. mais augmente la captation de glucose, l'oxydation du glucose et la liberation de lactate. Une diminution des triglyc6rides myocardiques, une augmentation du contenu en acides gras libres et de la liberation de glycerol, sans changements du contenu en cholest61-01 et en phospholipides, sont observtes apres augmentation de la concentration extracellulaire de calcium. Par rapport aux coeurs perfus& avec un milieu ctCpourvu de calcium, les taux de cl-Catine phosphate et d9ATI' sont moins ilevts alors que ceux d'ABP sont plus klevks seulement dans les coeurs perfusis avec un milieu contenant 5 mM de calcium. Aucune difference de contenu cn AMP et en A M P cyclique n'est relevee dans les coeurs perfuse.; h concentration diffkrente de calcium. L'activiti contractile augmente initialernent apr6s augmentation de la concentration de calcium de 1.25 2 5 m M et diminue ensuite jusqu'aux valeurs temoins. Les coeurs sont incapables de developper une force contractile en l'absence de calcium, alors que la force contractile diminue, puis revient A la normale sous l'effet d'une perfusion du coeur avec 0.31 mM de calcium. Ces rksultats indiquent que des valeurs ClevCes du calcium intracellulaire stimulent les processus glycogCnolytiques, glycolytiques et lipolytiques dans le mymarde. [Traduit par le journal] ABBREVIATION: CrP. creatine phosphate. 'This work was supported by a grant from the Manitoba Heart Foundation. 'Please address correspondence to: Dr. Naranjan S. Dhalla, Professor of Physiology, Fdculty of Medicine, University of Manitoba, Winnipeg, Man., Canada K3E OW3.
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Calcium is considered to play a crucial role in the regulation of celIular function and metabolism (Basmussen et a&. 1972). A great deal of effort has been made for understanding the participation of calcium ions in a wide variety of events intinaately associated with contractile activity of myocardium, and it is now established that calcium links the processes of excitation and contraction (Langer 1968; Sonnenblick and Stana I969 ;Katz 1978; Reuter 1974). However, relatively little is known about the involvement of calcium in processes regulating myocardial metabolism. Since calcium is known to increase the activity of myocardial lipase (EC 3.1.1.3) and phosphorylase kinase (EC 2.7.6.38) phosphorylase systems (Neely, Roveto et a&. 1972; Jesnnok et ak. 1976; Friesen et al. 1969; Gross and Mayer 1975), it is conceivable that this cation may participate in the processes of Iipolysis and glycogenolysis in myocardiuln. To gain some information in this regard the effects of calcium on the abilities of the isolated heart to use endogenous lipids and glycogen, and to prodaacc glycerol and lactate were tested. The abilities of tlae hearts to take up and oxidize glucose were also examined in the presence of different concentrations of calcium in the perfusion medium. The myocardial contents of cyclic AMP and adenine nucleotides were measured because changes in their concentrations are known to have profound effects on the glycolytic, glycogenolytic, and lipolytic activities of myocardium (Haugaard and Hess 1965; Hiraams-Hagen 1967; Wobison et al. 1968; Neely, Roveto et al. 8972). The contractile activity of these hearts was monitored during the course of perfusion with medium containing different concentrations of calcium.
Male hooded rats weighing 300-350 g were decapitated. the hearts quickly removed and arranged for coronary perfrision by the Langendorff technique as described earlier (DhalIa ef al. 1973). The KrebsHenseleit solution, pH 7.4, containing 8 m M glucose and different eonccntrations of calcium was gassed with a mixture of 95% O2and 5% C02, and maintained at 37°C. The hearts were punctmed with the sharp end of a pair of small scissors to avoid accumulation of fluid in the ventricles. A resting tension of 1 g was applied to the heart and the contractile force was
monitored on a Grass polygraph via a force displacement transducer QFT.03).All hearts were equilibrated with the perftisisn medium containing 1.25 mM calcium POP 15 min in an open system. At the end of this period the hearts were switched to a closed perfusion system (Bleehen and Fisher 1954) containing 50 ml of the desired medium. The coronary Wow was maintained at 10 ml/min with the help of a peristaltic pump. Perfusate (0.5-1 ml) was withdrawn for biochemical analysis at different intervals of the perfusion; this fluid was not replaced but appropriate corrections were made while calculating the data. At the end of each experiment, the hearts were frozen for biochemical detcrrninaltion with a bvollenberger clamp precooled in liquid N,. Dry weight of a portion of the ventricle was obtained by keeping the tissue in an oven at 100°C for 2 days. Glycogen from myocardium was extracted with hot 30% KOM and measured by the glucose oxidase (EC' 1.1.3.4) method (Huggett and Nixon 1957). Glucose uptake and lactate release from the isolated rat heart were measured by the procedures described by Williamson ( 1966). "CO, production was measured according to the method described by Kreisberg and Williamson (1964) upon perfusing the hearts with a medium containing 8 m M [kT-l'C]gTucsse (0.15 p C i / n ~ l( I Ci = 37 GBq)). Phospholipid and neutral lipids were extracted and separated by the method of Folch et al. ( 1 957) and Bsrgstrom ( 1952). The phospholipid phosphonns, triglyceride glycerol, cholesterol, and free fatty acids were measured according to the procedures descrikcd elsewhere (Wells and Dittmer 6965; Sperry iind Webb 1950; Rapport and AIonzo 1955). The ]high energy phosphate stores (CrP, ATP. ADP, and AMP) were determined by the flusrometric methods (Fcdelesova and Dhalla 1971 ). The extraction and measurement of cyclic AMP were carried out by the methods of Brown et ul. ( 197 1) and Cilman ( B970), respectively.
Jn one set of experiments, rat hearts were perfused for 40 min with medium containing 0, 0.31, 1.25, and 5 mM calcium; the concentxations of glucose or lactate in the perfusate and the production of d4CC02were measured at different intervals and myocardial glycogen contents were determined at the end of the perfusion period. The data shown in Figs. 1 and 2 indicate that the cardiac glucose uptake and lactate output were increased upon increasing the concentration of calcium in the perfusion medium. An increase in the oxidation of glucose was evident since an increase in 14@Oip production froin [U-14G]glucose was observed upon increasing the concentration of calcium in the perfusion medium (Fig. 3). Myocardial levels of glycogen declined markedly upon perfusing
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DI-IALLA ET AL.
TlME (min)
FIG.I . GIucose uptake by the isolated rat heart perfused with different concentrations of calcium for a period of 48 min. Each value is an average of five experiments. Vertical bars indicate 2 SEM.
FIG.2. Lactate output by the isolated rat heart perfused with different concentrations s f calcium far a period of 40 min. Each value is an average of six experiments. Vertical bars indicate 2 SEM.
the hearts with elevated levels of calcium (Fig. 4) The second set of experiments was performed to determine changes in lipid contents and the ability of heart to release glycercd upon perfusion with medium containing 0, 0.3 1, 1.25, and 5 mM concentrations of calcium. The data in Fig. 5 indicate that the level af triglycerides, but not that of cholesterol, in hearts perfused with medium containing 0.31-5 mM Ca2+was lower than that in the Ca2+-freeperfused hearts. On the other hand, thc concentration of free fatty acids, but not of phospholipids, was higher ( B < 0.05) in hearts perfused with 0.31-5 ntM ca?+in cornparison with that in hearts perfused with Ca"-free medium (Fig. 6 ) . The amounts of glycerol released from the hearts perf used with medium containing 0.31 -5 mM CaN were also higher than those from the hearts perfused with Ca2+-free medium (Fig. 7). Tn the third set of experiments, high energy phosphate stores and cyclic AMP concentrations in the hearts were measured after perfusion with different concentrations of calcium
for 40 min. The results in Table 1 reveal that CrP and ATP levels were lower and ABP levels higher in hearts perfused with 5 mM Ca2+ in comparison with hearts perfused with Ca2+-f ree medium. No change in AMP and cyclic AMP contents was seen in hearts upon perfusion with different concentrations of Caw. The time course of changes in contractile force due to perfusing the hearts with different concentrations of calcium is shown in Fig. 8. The hearts were unable to generate contractile forcc within 30 s to 1 min when perfused with Ca2+-free medium. It was interesting to note that the contractile force declined by about 75% within 2-4 min but thereafter started to recover towards the control levels over a period of 36 nlin when the hearts were perfused with 0.31 mM Ca9* Marked increments in the contractile force were seen upon perfusing the hearts with 2.5 or 5 mM Ca2+ within 2-4 rnin but thereafter started to decline towards the control leerel within $0 min of perfusion. A similar pattern of changes in the contractile force due to pelfusion with media containing
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TIME
(min)
F ~3. ~I .~ ~ production O , by isolated rat heart perfused with different concentrations of calcium for a period of 40 min. Each value is an averagc of four experiments. Vertical bars indicate 2 SEM.
1977
FIG.4. Glycogen levels in the isolated rat heart perfnsed with difTcrent concentrations of calcium for a period of 40 min. Each value is a mean & SE of I I
described here is a mean t SE of four to seven experiments. 'To determine if the observed effects sf calciu ~ narc mediated through the endogenous catecholamines - adrenergic receptor activation, ~ 0 1 1 1effects ~ of calcium were tested on hearts pcrfused with medium containing propranololl, a well-known P-adrenergic receptor blocking agent. It was found that perfusing hearts with 50-100 p M gropranolol, which completely blocked the positive inotrspic effect of 0.5 p g epinephrine, failed to alter the positive inotropic effect (68 t 3 % increase at 10 rnin), lactate production (26 1.7 pmol lactate/g dry heart weight per 10 rnin), and glycerol release (1.5 t 0.2 pmoI glycerol/g dry heart weight per 10 nmin) due to 5 mM Ca". The results described SE sf four experiments. here are mean
0-5 mM calcium was observed in hearts paced electrically at 250 beats/mimn at a voltage just above the threshold. Thus, it is unlikely that the observed changes in contractility are due to any alterations in heart rates. The time course of changes in myocardial metabolism due to 5 mM Ca" was also investigated. Switching the hearts to medium containing 5 mM Ca" was found to increase the contractile force by 79 t 0.9% of the control levels in 2 rnin without any significant changes in the coltcentrations of cyclic AMP, AMP, or ATP; however, the levels of ADP were increased by 21 -t- 0.5% of the control values. Significant reductions in the glycogen (25 31.3% decrease) and CrP (19 +- 1.5% decrease) contents were seen at 5 and 10 min after starting the perfusion with 5 mM CawC, Discussion respectively. whereas significant decreases in triglyceride (21 It 0.7% decrease) and ATP In this study we have shown that increasing (22 t 0.8% decrease) content were observed the concentration of calciunl in the perfusion after 20 rnin of pedusing the hearts with 5 mM medium resulted in decreasing triglyceride and ca2+ . It should be mentioned that each value glycogen contcnts of the heart. This lipolytic _+
IIHALLA ET AL.
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TRlGLYCERlDES
C
(pmol/o
k
CHOLESTEROL
dry
( p r n o ~ / ~d r y
FREE FATTY heart
vrrigkt )
h a a r t ueiph8 )
.I
I
.)
ACIDS
(.J J R R O I / Qd r y
( p r p p o ~ / ~d r y
heart
heart
weight
1
weigh?)
1 4
C ~ + C B N C L N T W A JfmM) IOM
FIG.5. Triglyceride m d cholesterol levels in the isolated rat heart perfused with different concermtrations of calcium for a period of 40 min. Each value is a mean zk SE of nine experiments.
effect of calciurn was also obvious from the elevated levels of free fatty acid without any changes in the phospholipid or cholesterol contents in myocardium as well as increased release of glycerol in the perfusion medium upon perfusing the hearts with increasing concentrations of calcium. Although the glycogenslytic effect of calcium was associated with an increased production of lactate by myocardium, glucose uptake was also increased upon elevating the concentrations of calcium in the perfusion medium. Raising the concentration of external calcium has been shown to enhance the permeability of glucose in skeletal muscle ( WolHoszy and Narahara 196'7 ) . The observed increase in glucose uptake may probably be due to the inlnibitory effect of calcium on the Na+-K+ ATPase, which has been considered to be intimately involved in regulating the transport of glucose across the cell membrane (Schwartz et al. f 972). On the other hand, the glycogenolytic and lipolytic effects of calcium are mediated due to the activation of phosphorylase and triglyceride lipase, respectively
FIG.6. Free fatty acid and phospholipid levels in the isolated rat heart perfused with different somentratisns of calcium for a period of 40 min. Each value is a mean 9 SE of nine cwperirnents.
(Friesen eir ah. 1969; Jesmok et a&. 1976). Decrease in glycogen and triglyceride contents, and increase in glucose uptake, g%ucose oxidation, glycerol release, arad lactate output by the isolated hearts have also been shown to occur in the presence of hormones such as epinephrine and glucagon (@orriblath et a!. 1963; \Vi8liamson 1964, 1966; Mrcisberg 1966; Kreisberg and Willian~strnf 964). These metabolic effects of hormones are elicited through the participation of cyclic AMP (Himms-Hagen 1967; Robison et ab. 19681. However, glycolytic, glycogenolytic, 2nd lipo'Lytic actions sf calcium cannot be considered to be mediated through cyclic AMP because its concentration did not change upon perfusing the hearts with elevated concentrations of calcium. Since perfusing the hearts with propranolol did not prevent the calcium-induced glycerol release or lactate production, It is unlikely that these metabolic effects of calcium are a consequence of norepinephrine release from the nerve endings present in the isolated heart preparations. Increasing the extracellular concentrat ion of calcium has been shoavw to increase myocardial
930
CAN. J. PEWSIBL. BHARMACOE. VOE. 55. 1977
TABLEI. High energy phosphate stores and cyclic AMP content of the isolated rat heart perfused for 40 min
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with mediun-i containing different concentrations of calcium Cowcn. of Catf in the perfusion medium, rnM
High energy phosphate cs~npoundsu,ymol/g dry heart weight CrP
AMP
ADP
ATP
Cyclic A M P prno8i.g dry heart weight
=Each value is a mean f- Sk; of sin experiments. bSisnifmcaratly different (P < 0.05) from the values obtniraed with Cal+-free (0 mM) medium.
P
GLYCEROL (ymol/g
RELEASED dry
heart
weight
1
FIG.7. Giycerol release from the isolated rat heart perfused with different concentrations of calcium for a period of 40 min. Each value is an average of nine experiments. Vertical bars indicate 2 SEM.
oxygen consumption (Sonnenblick et al. 1965) and this could be interpreted to reflect an increase in the processes involved in ATB production. This view is supported by our finding that an increase in l9CO2production from 1' 4Cjglucose was seen in the isolated hearts upon perfusion with elevated levels sf calcium. On the other hand, raising the external calcium is expected t~ increase rnyofibrillar ATBase activity
(Katz 1970) and result in an increased use of ATP. The rates of splitting of CrB and ATP in beating rabbit atria have been reported to increase upon increasing the concentration of calcium in the bathing medium (Schildberg and Fleckenstein 2 965 ) . The increased amounts of energy and ABP, which would be available due to increased hydrolysis of ATB, will be used for greater development of the contractile force
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DHAkLA ET A k .
0
8
16
24
32
QB
T l M E (rnin)
FIG.8. Contractile force of the isolated rat heart perfused with different concentrations of calcinm for a period of 40 min. Each value is an average of 25 experiments except that the values for 2.5 mM Ca2+are from four experiments. Vertical bars indicate 2 SEM.
and stimulating the mitochondria1 respiratory activity, respectively. Since increasing the concentration of calcium from 0 to 1.25 rmkl did not change the levels sf CrP or ATP in the perfused heart, it appears that ATP use due to elevated myofibrillar ATPase activity is balanced with ATP praduction due to an increased operation of rnitochondrial oxidative phosphorylation activity. It should be noted that both CrP and ATP levels declined in hearts perfused with medium containing 5 mM calcium. Such a change is most likely due to an insufficient process of ATB production, because under this condition myocardium is loaded with calcium (Tomlinson et wl. 1974) which may depress mitochondria1 oxidative ghosphorylation activity (Wrogernann and Pena 1976). At any rate, it is unlikely that the observed glycogenolytic, glycolytic, and lipslytic actions of calcium can be explained on the basis of changes in the concentrations of ATP, ABP, and AMP (Haugaard and Hess 1965; Neelly,
Benton et wl. 1972) since the levels of these nucleotides did not change upon perfusing the hearts with 0 to 1.25 rnkf calcium. A parallel relationship between external calcium and force of cardiac contraction has been clearly demonstrated (Loewi 1918; Luttgau and Niedergerke 6958). Such an increase in contractile tension by calcium has been suggested to be due to an increase in ATP use as well as conversion of chemical into mechanical energy (Furchgott and Lee I96 1) . However, in this study we have shown that the isolated hearts exhibit a remarkable ability of adaptation with respect to maintaining their contractile activity when perfused for prolonged periods with different concentrations sf calcium. For example, the contractile force decreased initially but recovered towards normal upon starting the perfusion with 0.31 mM calcium. Likewise, the contractile force increased initially but declined towards normal upon starting the perfusion with 2.5-5.0 mlM calci-
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CAN. J. PWSIOE. PHARMACOL.
urn. Whether these changes in mvocardiaI " contractility during the course sf perfusion with low or high concentrations sf calcium are due to some adjustments in the fluxes of calcium across different nsembranes or in the processes concerned witla energy production and use remain to be determined. It should be pointed out that increasing the extracellular concentration sf calcium has been shown to increase calcium influx (Winegrad and Shanes 1962) and this may elevate the intracellular level of calcium in the nsyocardial cell. Elevated levels sf the in-mtraicellularcalcium can also be assumed from the increased abilities of the hearts to generate higher contractile forcc tapon perfusion with high corpcentrations of extrace%lular caIcium. T~PUS, an increase irn the intracellular concentration of calciurn can be conceived to activate phosphorylase kinase directly and cause glycogenolysis (Gross and Mayer 1975). A similar kinapse syster-st has also been propsscd for the activation of the myocardial triglyceride lipase system by callcihan~(Sesmak et ab. 1974). The lipolytig: and g8ycogenolytic efFects of increased ventricular pressure (Crass et a/. 1969, 197l; Neely et a/, 1969; Neely, Denton sf aH. 19721, in which condition cab cium movements within the myocardiunn must be augmented, for increasi~sgcontractility can also be explained on the basis of calcium involvement. Since epinephrine is known to increase calcium influx (Remter 1974), some contribution of calcium in hormone-induced glycogenoIysis and Iipolysis in heart cannot be ruled out.
BHFEHEN, N . Me, and FISHER, R. B. 1954. The action of insulin in the isolated rat heart. J . Physiol. (London), 123, 260-274. B~MGSTROM, B. 1952. Investigation on lipid separation methods. Separation of phaspkolipids from neutral Pat and fatty acids. Acta Physiol. Scand. 21, 181-110. BROWN,B. L., ALBANO,J . D. M.,Earn~s,R. P., and SGIIEWZH. A. M. 1971. A simple and sensitive saturation assay method for the measurement of adenosine3':s'-cyclic naonophosphate. Biochem. J. 121, 561562. CORNWLATH, M., RANDLE, P. J., PARMEGGIANI, A., and MORGAN. H. E. 1963. ReguHa tion of glyccsgenolysis in rnuscIe: effect of giucagon and anoxia on lactate producta'sn. glycogen content and phosphorylase activity in the perfused rat heart. J . Biol. Chern. 233, 1592-1 597. CRASS,IPI, M. F., MCCASKELL, E. S., and Snirp~,J. C. 1969. Effects of pressure development on glucose
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and palmitate metabolism in perfused heart. Am. J. Physiol. 216, 1569-1 576. CRASS.111, M. F., M@CASKEI,E, E. S., SHIPP,%.C . , and MURTHY,$7. K. 1971. Metabolism of endogenous lipids in cardiac mtlscle: Effect of pressure development. Am. J. PhysioI. 228,428-435. DHAELA, N. S., MATOUSEIEK, R . F., SUN, C. N., and BESON,R. E. 1973. hfetabelic, ultrastructural and rnechanicaB changes in the isolated rat heart perfused with aerobic medium in the absence or presence of glucose. Can. J. PhysioI. Pharmacol. Sf. 590-603. FEDEIESOVA, M., and DIIALLA, N. S. 1971. High energy phosphate stores in the hearts of genetically dystrophic han-ssters. %.Mol. Cell. CarclioB. 3, 93-182. Fo~c-11, J., 1 ~ ~M., s and . SEOAN-STANILY, G. W. 1954. A simple method for the isolation and purification of total lipids from animal tissues. J . Niol. Chem. 226,497-589. FRICSEN,A. 3. D., OLIVER,W., and ALLEN,G . 1969. Activation of cardiac glycogen phosphorylase by calcium. Am. J. Physiol. 217, 445-450. FURCPI~OTT, R. F., and LEE,,K. S. 1961. Nigh energy phosphate and the force of contraction of cardiac muscle. Circulation, 24, 416-428. GII-MAN,A. G . 1940. A protein binding assay for adenosine 3"5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67, 305-3 12. Claoss, S. W., and MAYER,S. E. 1975. Phosphorylase kinase mediating the effects of cyclic AMP in rnrascle. Metabolism, 24, 369-380. HAUGAARD, N., and HESS, M. E. 1965. Actions of autonomic dmgs on phssphorylase activity and function. Pharrnaeol. Rev. 17, 27-70. HIMMS-MAGEN, 9. 8967. Sympathetic regulation of rnetabolisrn. Pharmacol. Rev. 19, 367-461. Moe~oszu.J. 0., and NARAHARA, £3. T. 1967. Enhanced perrneztbi8ity to sugar associated with muscle contraction. Studies sf the role s f Cab-. I . Cen. Physiol. 568. 55 1-562. HUGGET, A. St. G., and NIXON,D. A. 1957. Use of glucose oxidase, peroxidase and 0-dianisidine in determination of blood and urinary glucose. 1,ancet ii, 368-370. JESMOK, G. J . , MOGELNICKI, S. W., LECH,J. J., and CAIVERT, D. N. 1976. Effect of polyamines, ionc and epinephrine on triolein hydrolyzing activity of rat venbricallar extracts. J. Mol. Cell. Cardiol. 8, 283-298. KATZ,A. M. 1970. ConItractiIe proteins of the heart. Physiol. Rev. 50, 63-158. KREISBERG, R. A. 1966. Effect of epinephrine on n-syocardial triglyceride and free fatty acid utilization. Am. J, Physiol. 210, 385-389. KREISBERG, R. A.. and WILLIAMSON, J. R. 1964. Metabolic effects of glucagon in the perfused rat heart. Am. J. Physiol. 207, 721-727. LANGLR, G. A. 1968~Ion fluxes in cardiac excitation and contraction and their relation to myocardial contractility. Physiol. Rev. 48,708-757. I[,oE.wI, 0. 19 18. Uber den Zusammenhang Zwischen Digitalis und KaIziumwirkung. Arch. Exp. Patkol. Pharrnak. 882, 131-1-98.
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