Cardiology. Basic Res Cardio187:139-147 (1992). Adrenergic stimulation of rat hearts with severely reduced cytosolic adenine nucleotide pool and [ATP]/[ADP] ...
Basic Research in
Cardiology
Basic Res Cardio187:139-147 (1992)
Adrenergic stimulation of rat hearts with severely reduced cytosolic adenine nucleotide pool and [ATP]/[ADP] ratio V. V. Kupriyanov, O. V. Korchazhkina, V. L. Lakomkin, and V. I. Kapelko Institute of Experimental Cardiology, Russian Cardiology Research Center, Academy of Medical Sciences, Moscow, Russia
Summary: The effect of severe reduction of cytosolic adenine nucleotide (AdN) pool and [ATP]/ [ADP] ratio (by 2-deoxyglucose treatment) on functional and metabolic responses of isovolumic rat heart to increased energy demand induced by coronary flow (CF) rise and isoproterenol (Iso) addition has been investigated, AdN-depleted hearts had reduced phosphocreatine (PCr, by 80 %), ATP (by 75 %), [ATP]/[ADP] (24 times) and pressure-rate product (PRP, by 60%). An elevation of CF was followed by the increase in PRP in control and AdN-depleted hearts by 40-45 % with unchanged metabolic parameters. At increased CF, Iso caused a further rise in PRP in both groups due to elevation of heart rate; however maximal levels of PRP in the AdN-depleted group still remained lower than that of control (by 40 %). Only in control experiments was Iso addition accompanied by an increase in the difference between left-vetricular end- and minimal diastolic pressure, cytosolic [Pi] and [ADP], and some decrease in PCr and [ATP]/[ADP]. These data imply that severely reduced eytosolic [ATP]/ [ADP] does not prevent acceleration of Ca2+ turnover by Iso in cardiomyocytes, it but restricts maximal force development affecting the myofibfils. Key words: [3-adrenergic stimulation; cardiac work; [ATP]/[ADP] ratio; a_denylate depletion; 3~p_ NMR, rat heart Introduction Limitations in ATP supply to myofibrils and ionic pumps in cardiomyocytes are usually associated with both suppression of contractile function, and with reduced and transient response to adrenergic stimulation (26). In fact, these events take place during anoxia or metabolic inhibition of mitochondria (26) and creatine kinase (14), and are related to decreases in cytosolic [ATP]/[ADP] and ATP affinities (A(ATP) = - AG(ATP)) (14, 26). However, moderate inhibition of the respiratory chain with amytal did not decrease the degree of stimulation of cardiac work and oxygen consumption rate by isoproterenol, despite reduced A(ATP) (16). These observations raise the question of whether responsiveness of hearts to catecholamines can be restricted by decreased cytosolic [ATP]/[ADP] or A(ATP). Earlier, we (15) and Hoerter et al. (10) found that perfusion of well-oxygenated rat hearts with 2-deoxy-D-glucose (DG) in the presence of pyruvate (15) (as well as acetate (10)) resulted in severe depletion of the cytosolic pool of adenine nucleotides, and in a decline in [ATP]/[ADP] ratio. This was due to phosphate trapping into 2-deoxyglucose-6phosphate, but not due to mitochondrial dysfunction (10, 15). For this reason, this model is suitable for assessment of the role of [ATP]/[ADP] ratio in the control of contractile function and its response to [3-adrenergic stimulation. In this study, we found that severely reduced cytosolic [ATP]/[ADP] ratio in perfused rat heart did not prevent isoproterenol-induced elevation of cardiac work index. This implies 716
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t h a t activation of C a 2+ t u r n o v e r by c a t e c h o l a m i n e s is capable of o v e r c o m i n g the inhibitory effect of u n f a v o r a b l e t h e r m o d y n a m i c conditions o n contractility. Materials and Methods
Perfusion of isolated heart preparations Hearts of male Wistar rats weighing 250-350 g were perfused by the Langendorff method with Pi-free Krebs-Henseleit buffer containing 5 mM pyruvate instead of glucose. A water-filled latex balloon was placed in the left-ventricular (LV) cavity and LV pressure, as well as its first derivatives, were measured by a pressure transducer (Gould Statham P23Db) and recorded (Gould 2400 recorder). Coronary flow rates were provided by a peristaltic pump (Masterflex) and, hence, were independent variables, whereas perfusion pressure was a function of coronary flow. The pressure-rate product (PRP) was calculated as the product of LV developed pressure (LVDP) and spontaneous heart rate (HR). To introduce the perfused heart into the wide-bore magnet of the NMR spectrometer (CXP-200, Bruker) the heart was placed in a rubber sack, and perfusate was kept at a level that provided complete immersion of the heart. The rubber sack with the heart inside was placed in a 20 mm sample tube containing D20, and this system was positioned in the NMR magnet (18). Experimental protocol. The protocol is shown below. Time min, Steps Coronary flow
30-40
30
CF1
Additions
10
10
10
5
20
I
II
III
IV
V
CF2
CF1
DG insulin
Iso
After a 30- to 40-rain stabilization period hearts were perfused for 30 rain with 2 mM 2-deoxy-Dglucose (DG) and i0 IU/L of insulin added to the standard perfusion solution described above. Then, insulin was washed out during 10 min and, afterwards, coronary flow was elevated from 63.4_+ 2.5 (CF1) to 97.2_+3.4 ml/min-g dry wt (CF2) for 10 min. It was followed by addition of 0.1 btM isoproterenol (Iso) for 10 rain and its subsequent 5-rain washout. DG (2 mM) was present during all of these treatments (35 min) in order to compensate for dephosphorylation of 2-deoxyglucose-6-phosphate and, hence, recovery of metabolic and functional variables that usually takes place in the absence of D G (15). Finally, coronary flow was returned to the initial value and perfusion continued for 20 rain. Control hearts were perfused according to the same protocol, but without additions of DG and insulin. At the end of the experiments hearts were frozen in liquid nitrogen and used for biochemical assays as described below.
N M R experiments 3lp-NMR spectra of perfused hearts were usually accumulated each 5 min at standard frequency 80.98 MHz by applying 90 ~ (37 Ds) sampling pulses with 2 s repetition time. Each spectrum was obtained by collection of 150 transients in a 4K memory block; the resulting signal was exponentially multiplied with the line-broadening factor 10-20 Hz and was Fourier transformed, which yielded a frequency spectrum in the 4000 Hz ( - 5 0 ppm) spectral range. To suppress spin-spin interaction (IH-31P) broadband proton decoupling (0.2 watt) was used over the period of spectra acquisition time. At the beginning and at the end of the experiment, "quantitative" spectra with 10s repetition time were accumulated. Peak areas cut off from these spectra were used to calculate tissue content of PCr, ATP, Pi, and DG-6P. Peak of methylene diphosphonate (-- 10 ~moles of 31p) was used as reference for spectra quantitation. A solution (100 mM) of this compound sealed in a plastic tube was placed in the vicinity of the heart.
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Biochemical assays Frozen tissue was pulverized in a mortar and extracted with 6 % cold perchloric acid with 10 % methanol. ]Neutralized extracts were used for total creatine assays by the method of Eggleton et al. (6). Calculations of concentrations of cytosolic phosphates Concentrations of PCr and Pi were estimated by assuming cytosolic water content to be 2.5 ml/g dry tissue (3). Pi was considered as completely cytosolic, since a chemical shift of its peak corresponded to cytosolic pH 7.1-7.2. In normal hearts, cytosolic ATP was taken to be 85 % of the total, assuming that 15 % is mitochondrial ATP (7, 19), which is visible in 31p-NMR spectra. It is implied that mitochondrial ATP (and AdN) cannot be depleted with DG due to the following reasons: 1) hexokinase phosphorylating DG is localized out of mitochondrial matrix; 2) DG cannot penetrate across the inner mitochondrial
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Fig. 1. Time-course of LV systolic (A) and diastolic pressures (B) and perfusion pressure (C) in control and DG-treated hearts during the experiment. LVSP, EDP (circles), MDP (triangles), and PP are leftventricular systolic, end-diastolic, and minimal diastolic pressures, and perfusion pressure, respectively. Open symbols refer to control hearts perfused with 5 mM pyruvate and without 2-deoxyglucose (DG) treatment. Closed symbols refer to the hearts treated with 2 mM DG and 10 IU/L of insulin during 30 rain (from 0 to 30 rain). At 30 min, insulin was removed and a standard protocol that included different loads (steps I-IV, see Materials and Methods) was followed. Iso, isoproterenol, 0.1 ~tM. CF1 and CF2 are coronary flows provided by peristaltic pump and equal to 63.4 4--2.5 and 97.2 +_3.4 ml/min, g dry wt, respectively.
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membrane, and 3) under normal conditions mitochondrial ATP is exchanged for cytosolic A D P in stoichiometry 1:1, which assumes constancy of mitochondrial pool of AdN. This theoretical consideration was confirmed in our recent work (17), in which we have estimated the fraction of cytosolic AdN using saponin treatment. Mitochondrial and myofibrillar A d N were not affected by D G treatment and comprised about 25 % of the normal total content, in accordance with earlier observations (7, 19). Cytoplasmic [ATP]/[ADP] ratio was calculated from creatine kinase equilibrium by the formula: [ATP]/[ADP] = Keq' [PCr]I[Cr]. Equilibrium constant (Keq) was taken to be 104, assuming cytosolic p H 7.2 and [Mg2+]free = 1 mM (20). Free creatine was estimated as a difference between total [Cr] and [PCr]. Free [ADP] and A(ATP) = - A G ( A T P ) were calculated by using [ATP]/[ADP] ratio and cytosolic [ATP] and [Pi] according to: A(ATP) = -AGo + 2.3RT. log([ATP]/[ADP] [Pil), where AGo = - 3 0 . 5 kJ/mol (8).
Reagents Sodium pyruvate, glucose, enzymes, nucleotides, dithiothreitol, insulin, and isoproterenol were from Sigma (USA). 2-Deoxy-D-glucose and E D T A were supplied by Merck (FRG). Salts were of analytical and purest grades.
Table 1. The effect of isoproterenol on functional parameters of normal and AdN-depleted hearts. Parameter
Group
Steps II
I
63.6
IIl
Coronary flow, ml/min 9g dry wt 97.2 97.2 + Iso
V
63.6
PRP, m Hg/min
Control DG
34.6 • 1.9 13.1_+2.4
49.0 • 5.4* 1%0_+1.9*
58.8 • 4.4* 33.5+3.5*
25.3 _+ 3.4 10.8+2.7
LVDP, mm Hg
Control DG
155 • 17 74 _+ 18
189 • 20 96 + 8
164 _+ 13 96 • 12
107 _+ 15 51 • 3
HR, min 1
Control DG
210 • 12 192 • 24
258 _+ 18 198 • 18
360 • 6* 348 • 8*
240 -+ 24 214 _+ 12
L V E D P - LVMDP, mm Hg
Control DG
4.7 • 0.9 1.2 • 0.6
4.8 _+3.2 1.6 • 0.89
26.6 _+ 7.1" 2.8 _+2.8
3.3 • 1.9 0.8 • 0.59
(+dP/dtm)/LVDP, s -1
Control DG
21.5 _+ 2.54 19.0 • 1.12
22.8 • 2.44 18.3 • 1.81
25.8 • 2.39 22.1 • 1.71
19.2 • 1.81 15.7 _+2.07
(-dP/dtm)/LVDP, s -1
Control DG
10.1 + 0.66 9.7 • 1.23
10.9 • 0.35 10.6 • 0.61
18.7 • 1.75" 15.1 • 0.75*
12.4 _+ 0.51 11.3 • 0.34
Means + SE are given for five hearts in each group. PRP, pressure-rate product; HR, heart rate; LVDP, LV developed pressure, is the difference between systolic and end diastolic pressures; L V E D P LVMDP, difference between end - and minimal diastolic pressures; (+dP/dtm)/LVDP and (-dP/dtm)/ LVDP are the ratios of maximal rates of contraction and relaxation to LVDP, which are indices of contraction and relaxation, respectively. Steps I to V are as described in Experimental protocol (see "Methods"). lsoproterenol concentration, 0.1 ~tM. Data for all steps were taken between 5 and 10 min after changes when new steady-state was established. Asterisks denote statistically significant difference as compared with respective initial values. Difference in PRP and LVDP values between two groups are statistically significant for all steps (p < 0.01).
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Results
Functional changes Figure 1 and Table 1 demonstrate effects of coronary flow increase and Iso addition on functional parameters of normal and AdN-depleted rat hearts. The latter were characterized by significantly reduced PRP due to decreased LVSP (by 80 mm Hg, Fig. 1) and elevated end~diastolic pressure (by 18 m m H g , Fig. 1) while spontaneous heart rate (HR) remained close to the control. Minimal diastolic pressure (MDP) rose after D G treatment as much as EDP (by 20 m m H g , Fig. 1). Also, the maximal rates of LV pressure development (+dP/ dtm) and decay (-dP/dtm) were declined proportional to the decrease in developed pressure, so that their ratio to LVDP (contraction and relaxation indexes) remained constant. Elevation of coronary flow rate and perfusion pressure 1.5 times (from 75-85 to 112-127 mm Hg, Fig. 1) resulted in proportional augmentation of the PRP value, due to an increase in LVDP in both groups, and also due to an increase of HR in the control group only (Table
A
PCr ATP
DG- 6P
20
10
0 ppm
-10
-20
Fig. 2. Representative 3~p-NMR spectra of perfuscd rat heart before (A) and after (B) depletion of adenine nucleotides. R, DG-6P, and PCr are peaks of reference (methylene diphosphonate), 2-deoxyD-glucose-6-phosphate and phosphocreatine. Spectra are the sum of 150 acquisitions with 2-s repetition time. Line-broadening factor during exponential multiplication is 15 Hz. Chemical shifts are given with respect to PCr peak. The heart (0.19 g of dry wt) was perfused with Krebs-Henseleit buffer containing 5 mM pyruvate-Na as oxidizable substrate (A), and then 2 mM 2-deoxy-D-glucose with 10 IU/L of insulin were added and perfusion lasted 30 min (B).
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1). In contrast, there was no change in the difference between L V end-diastolic and minimal diastolic pressures, or in the ratios of +dP/dtm and -dP/dtm to L V D P . Administration of isoproterenol at increased coronary flow was followed by further significant elevation of P R P values in both groups and was predominantly due to an increase in heart rate; the effect was even m o r e pronounced in A d N - d e p l e t e d group (75 % vs 20 % in control; Table 1). Simultaneously, the relaxation index considerably increased in both cases, however, this increase was less in the A d N - d e p l e t e d group. The difference between L V enddiastolic and minimal diastolic pressures rose only in the control group, due to decrease in M D P (Fig. 1). Changes induced by Iso and coronary flow elevation were reversed (although not completely) when initial conditions were re-established (Fig. 1 and Table 1). It is interesting that both groups had similar coronary resistance (ratio of PP to CF) which was not affected by Iso.
Metabolic changes Figure 2 shows representative spectra of control and A d N - d e p l e t e d heart, it is seen from the Figure and Table 2 that, besides severely reduced total A T P content, A d N - d e p l e t e d hearts had much lower concentrations of PCr and Pi, as well as [ATP]/[ADP] ratio, A ( A T P ) (by 6 kJ/mol), and a somewhat higher free [ADP]. Table 2 shows alterations in cardiac cytosolic phosphates linked to functional effects described above. Elevation of coronary flow did not alter all these parameters in either group; however, the response to adrenergic stimulation was different. In the control group, Iso decreased PCr and A T P concentrations by 25 % and 20 %, respectively, and concomitantly increased [Pi] by 65 %. This resulted in a doubling of free A D P concentration, and a reduction of [ATP]/[ADP] and A ( A T P ) . In Table 2. The effect of isoproterenol on metabolic parameters of normal and AdN-depleted hearts. Parameter
Group I
63.6
II
Steps III
Coronary flow, ml/min - g dry wt 97.2 97.2 + Iso
V
63.6
PCr, mM
Control DG
16.8 _+0.6 3.0 _+0.49
17.1 + 0.29 2.88 + 0.47
12.8 _+ 1.0" 3.75 _+0.63
15.2 _+0.58 4.12 _+0.40
Pi, mM
Control DG
5.32 + 0.96 2.24 _+0.72
5.34 + 1.06 2.46 _+0.81
8.8 _+2.16" 2.80 • 0.81
5.44 _+ 1.44 2.84 _+0.79
ATPtot, mM
Control DG
6.12 • 0.80 1.54 • 0.21
6.32 _+0.70 1.45 _+0.15
4.96 _+0.88 1.45 _+0.15
4.76 • 0.56 1.45 _+0.14
ADP, vM
Control DG
16.2_+3.0 29.0 _+5.1
14.5_+1.7 26.3 _+4.9
29.8_+5.1" 17.3 _+3.3
17.8_+2.2 17.1 _+2.4
[ATP] / [ADP]
Control DG
367 _+65 15.6 _+3.3
387 _+61 14.7 _+2.9
174 _+48* 21.3 + 5.6
262 _+58 21.8 _+4.6
A(ATP), kJ/mol
Control DG
59.2 _+0.7 53.4 _+ 1.2
59.5 _+0.7 53.2 _+ 1.1
55.9 _+0.9* 53.6 _+ 1.1
58.7 _+ 1.2 53.7 _+0.8
Means _+SE are shown for five hearts in each group. Cytosolic concentrations of phosphates, [ATP]/ [ADP] and A(ATP) were calculated as described in Methods. Perfusion steps correspond to those are given in Methods and in the legend to Table 1. Asterisks denote statistically significant difference as compared with the initial values. Differences in all parameters between two groups are statistically significant for all steps (p < 0.01).
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145
contrast, these parameters did not change in AdN-depleted hearts. Removal of Iso and resumption of the initial coronary flow fully restored initial values of the parameters in control group, except for ATP levels, which remained reduced by 20 %. Intracellular pH did not show a detectable change during isoproterenol infusion in either group and was in the range of 7.1-7.2, as estimated from the position of the Pi peak in NMR spectra.
Discussion
Data obtained in this study concern a possible role of cytosolic phosphates in the regulation of cardiac contractile function and their interplay with Ca a+ regulating systems. Decrease in cardiac work index and elevation of end-LV-diastolic pressure observed after DG treatment can beascribed to a severe fall in cytosolic [ATP]/[ADP] ratio and reduction of A(ATP). Their effects could be realized in two ways. First, a decrease in these parameters could slow down crossbridge turnover and increase the proportion of actin-bound myosin heads (23, 24). Second, this could lead to partial reversal of ionic pumps (Na,K-ATPase, Ca2+-ATPase of sarcoplasmic reticulum and sarcolemma) and, therefore, increase diastolic [Ca~+]. Note that, unlike hypoxia and metabolic inhibition which cause a simultaneous fall of [ATP]/[ADP], the increase in cytosolic Pi concentration (1, 14, 16, 25) in AdN-depleted hearts [Pi] was reduced (see Table 2). Hence, Pi, known as a potential inhibitor of contraction (14, 24, 25), could not contribute to suppression of cardiac work. Also, high concentration of 2-deoxyglucose-6-phosphate accumulated during DG treatment (ca. 25 raM) can hardly be an inhibitor of contraction and Ca 2+ turnover (15). Finally, intracellular pH and free Mg 2+ concentration did not change after D G treatment. Preservation of adrenergic stimulation of cardiac work in AdN-depleted hearts implies that, even at a very low [ATP]/[ADP] ratio, the myofibrils are capable of increasing their work and, perhaps, the rate of ATP hydrolysis. However, maximal levels of work performed by these hearts were significantly restricted because of lower LV developed pressure, as compared with the control group (see Table 1). This may mean that Ca 2+ turnover was inhibited by low [ATP]/[ADP] and A(ATP), and Iso released this process from inhibition via the known mechanisms of catecholamine action (11). A similar situation probably occurred when [ATP]/[ADP] was reduced by inhibition of the proximal end of the respiratory chain by amytal (16). Limitation of maximal performance can instead be related to restricted capability of myofibrils to develop maximal force due to reduced [ATP]/[ADP] and/or A(ATP) rather than to incomplete activation of Ca 2+ turnover by Iso. Lack of changes in steady-state concentrations of PCr, ADP, and Pi during isoproterenol action in AdN-depleted hearts implies coordinated stimulation of contraction and oxidative phosphorlyation by Ca ions or by some other factors. In spite of high concentration of pyruvate (5 raM) which should prevent activation of pyruvate dehydrogenase with Ca 2+ (22), this ion could activate two other dehydrogenases of Krebs cycle; isocitrate and 2-oxoglutarate DH, thus increasing mitochondrial [NADH]/[NAD +] ratio (5, 9, 12). Also, Ca 2+ activation of mitochondrial ATP-synthase has been suggested (4). Note that oxygen supply was fixed, since constant coronary flow was maintained by a peristaltic pump. In contrast, in control hearts an activation of ATP supply and utilization with Iso was not fully coordinated and was followed by an increase in eytosolic [ADP] and [Pi] (see Table 2). This response is characteristic for isovolumic hearts peffused with salt solution (2, 21). First, this could be due to higher maximal energy demands of myofibrils and lower energy supply by mitochondria, both provided by higher cytosolic [ATP]/[ADP] ratio as compared with that in AdN-deficient hearts. Second, inadequate oxygen supply into the tissue due to restricted coronary flow in conditions of increased energy demand might be another reason for such an
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imbalance. However, the latter seems to be less possible, since we did not observe signs of hypoxia, or acidification of intracellular medium. Unlike the response of cardiac work to {3-adrenergic stimulation that was qualitatively similar in both groups, the cardiac relaxation was changed in a different manner. In fact, in control groups Iso increased the difference between LV end-diastolic and minimal diastolic pressures that is the usual response to catecholamines (11), whereas in the AdN-depleted group this parameter (reflecting LV distensibility) was not altered. Also, (-dP/dt,~)/LVDP ratio (analog of relaxation index) increased markedly by Iso in both groups (see Table 1). The latter observation suggests that catecholamine-induced acceleration of Ca 2+ removal from myofibrils is not impaired in depleted hearts, despite dramatically decreased [ATP]/ [ADP] ratio. In summary, we may conclude that cytosolic [ATP]/[ADP] ratio and A ( A T P ) cannot be considered as major determinants of contraction and relaxation of cardiac muscle. However, lower values of these metabolic variables limit maximal cardiac performance that is induced by mechanical (coronary flow) and inotropic ([3-adrenergic) loads. The latter is associated with abolished Iso-induced increase in LV distensibility.
References
1. Alien DG, Orchard CH (1987) Myocardial contractile function during ischemia and hypoxia. Circ Res 60:153-168 2. Bittl JA, Balschi JA, Ingwall JS (1987) Effect of norepinephrine infusion on myocardial high energy phosphate content and turnover in the living heart. J Clin Invest 79:1852-1859 3. Bunger R, SobotI S (1986) Cytosolic adenylates and adenosine release in perfused working heart. Eur J Biochem 159:203-213 4. Das AM, Harris DA (1990) Control of myocardial ATP synthase in heart cells: inactive to active transitions caused by beating or positive inotropic agents. Cardiovasc Res 24:411--417 5. Denton RM, McCormack JG (1980) On the role of the calcium transport cycle in heart and other mammalian mitochondria. FEBS Lett 119:1-8 6. Eggleton P, Elsden SL, Gough N (1943) The estimation of phosphocreatine and diacetyl. Biochem J 337:526529 7. Geisbuhler T, Altschuld RA, Trewyn RW, Anzel AZ, Lamka K, Brierley GP (1984) Adenine nucleotide metabolism and compartmentalization in isolated adult rat heart cells. Circ Res 54:536-546 8. Griese M, Perlitz V, Jungling E, Kammermeier H (1988) Myocardial performance and free energy of ATP hydrolysis in isolated rat hearts during graded hypoxia, reoxygenation and high K + perfusion. J Mol Cell Cardiol 20:1189-1201 9. Hansford RG (1985) Relation between mitochondrial calcium transport and control of energy metabolism. Rev Physiol Biochem Pharmacol 102:1-62 10. Hoerter JA, Lauer C, Vassort G, Gueron M (1988) Sustained function of normoxic hearts depleted in ATP and phosphocreatine: a 31P-NMR study (1988) Am J Physiol 255:C192-C201 11. Katz A (1979) Role of contractile proteins and sarcoplasmic reticulum in response of heart to catecholamines. Adv Cyc Nuc Res 11:303-343 12. Katz LA, Koretsky AP, Balaban RS (1988) Activation of dehydrogenase activity and cardiac respiration: a 31p-NMR study. Am J Physiol 255:H185-188 13. Kentish JC (1986) The effect of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle. J Physiol 370:585-604 14. Kupriyanov VV, Lakomkin VL, Kapelko VI, Saks VA (1990) Myoplasmic phosphate metabolites in the integration of oxidative phosphorylation and contractile function in the myocardium. Biomed Sci 1:113-121 15. Kupriyanov VV, Lakomkin VL, Kapelko VI, Steinschneider AY, Ruuge EK, Saks VA (1987) Dissociation of adenosine triphosphate level and contractile function in isovolumic hearts perfused with 2-deoxyglucose. J Mot Cell Cardiol 19:729-740
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16. Kupriyanov VV, Lakomkin VL, Korchazhkina OV, Stepanov VA, Steinschneider AY, Kapelko VI (1991) Cardiac contractile function, oxygen consumption rate and cytosolic phosphates during inhibition of electron flux by amytal - a31P-NMR study. Biochim Biophys Acta 1058:386-399 17. Kupriyanov VV, Lakomkin VL, Steinschneider AY, Veksler VI, Korchazhkina OV, Kapelko VI, Saks VA (1991) Adrenergic stimulation of the heart during inhibition of phosphocreatine and adenylate pathways of energy transfer. Physiol J (Russ) in press 18. Kupriyanov VV, Steinschneider AY, Ruuge EK, Kapelko VI, Zueva MY, Lakomkin VL, Smirnov VN, Saks VA (1984) Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart. Biochim Biophys Acta 805:319-331 19. LaNoue KF, Bryla J, Williamson JR (1972) Feedback interaction in the control of citric acid cycle activity in rat heart mitochondria. J Biol Chem 247:667-674 20. Lawson JWR, Veech RL (1979) Effects of pH and flee Mg2+ on the Keq of the creatine kinase reaction and other phosphate transfer reactions. J Biol Chem 254:6528-6537 21. Matthews PM, Williams RA, Seymour A-M, Schwartz A, Dube G, Gadian DG, Radda GK (1982) A 31p-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain and ionophore RO2-2985 (X537A) in the isolated, perfused rat heart. Biochim Biophys Acta 720:163-1.71 22. Mekhfi H, Ventura-Clapier R (1988) Dependence upon high energy phosphates of the effects of inorganic phosphate on contractile properties in chemically skinned rat cardiac fibers. Pfltigers Arch 411:378-385 23. McCormack JG, England PJ (1983) Ruthenium red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart. Biochem J 214:581-585 24. Schoenberg M, Eisenberg E (1987) ADP binding to myosin cross-bridges and its effect on crossbridge detachment rate constants. J Gen Physiol 89:905-920 25. Sleep J, Glyn H (1986) Inhibition of myofibrillar and actomyosin subfragment 1 adenosine triphosphatase by adenosine 5'-diphosphate, pyrophosphate, and adenylyL5'-yl imidodiphosphate. Biochemistry 25:1149-1154 26. Williamson JR, Schaffer SW, Scarpa A, Safer B (1974) Investigation of calcium cycle in perfused rat and frog hearts. In: Dhalla NS (ed) Recent Advances in Studies on Cardiac Structure and Metabolism vol 4. University Park Press, Baltimore, pp 375-392 Received November 6, 1991 accepted January 20, 1992 Authors' address Dr. V. V. Kupriyanov, Inst. of Exptl. Cardiol., Cardiol. Res. Center, 3rd Cherepkovskaya str., 15A, Moscow, 121552, Russia
