AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H2192-H2200, 2004. First published June 24, 2004; doi:10.1152/ajpheart.00327.2004
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/H2192    most recent
00327.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Penna, C.
Right arrow Articles by Mavelli, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Penna, C.
Right arrow Articles by Mavelli, I.

F0F1 ATP synthase activity is differently modulated by coronary reactive hyperemia before and after ischemic preconditioning in the goat

Claudia Penna,1 Pasquale Pagliaro,1 Raffaella Rastaldo,1 Francesca Di Pancrazio,2 Giovanna Lippe,2 Donatella Gattullo,1 Daniele Mancardi,1 Michele Samaja,3 Gianni Losano,1 and Irene Mavelli2

1Sezione di Fisiologia, Dipartimento di Neuroscienze and Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, 10100 Turin; 2Dipartimento di Scienze e Tecnologie Biomediche and Centro di Eccellenza Microgravity, Aging, Training, and Immobility, Università di Udine, 33100 Udine; and 3Dipartimento di Medicina, Chirurgia, e Odontoiatria, Università di Milano, 20100 Milan, Italy

Submitted 6 April 2004 ; accepted in final form 22 June 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The amplitude of coronary reactive hyperemia (CRH), elicited by 15 s of ischemia, is reduced in hearts subjected to 5 min of ischemic preconditioning (IP). F0F1 ATP synthase activity and ATP concentration are also altered by IP. We hypothesized that F0F1 ATP synthase is differently modulated by the inhibitor protein IF1 during CRH elicited before (CRHnp) and after (CRHprec) IP. Hemodynamic parameters were recorded in 10 anesthetized goats. Myocardial biopsies were obtained before IP (Cnp), during CRHnp, 4 and 6 min after the onset of CRHnp, after IP (Cprec), during CRHprec, and 4 min after CRHprec. F0F1 ATP synthase activity, ATP concentration, and ATP-to-ADP ratio (ATP/ADP) were determined. Compared with CRHnp, IP blunted CRHprec. F0F1 ATP synthase activity transiently increased during CRHnp, decreased 4 min after CRHnp, and returned to control 2 min later; it was lower after IP (Cprec) and did not change during and after CRHprec. All these changes in activity were modulated by IF1. During CRHnp, ATP concentration and ATP/ADP were reduced compared with Cnp and began to rise 6 min thereafter. During Cprec, both parameters were transiently reduced but increased during and after CRHprec. Hence, during CRHnp, F0F1 ATP synthase activity transiently increases and then decreases significantly. The short-lasting inhibition of the enzyme may explain why a few seconds of occlusion do not induce IP. After IP, F0F1 ATP synthase activity is blunted, and it is not affected by a subsequent 15 s of occlusion, which induces a blunted CRHprec. These results suggest that postischemic long-lasting inhibition of F0F1 ATP synthase activity may be a feature of the preconditioned heart. The increase in ATP concentration after preconditioning is in agreement with previous reports of reduced ATP hydrolysis by cytoplasmic ATPases.

coronary circulation; energy metabolism; ischemia; mitochondria

THE IMMEDIATE SOURCE OF ENERGY for myocardial contraction is provided by the hydrolysis of ATP. Although ATP hydrolysis is carried out by a number of ATPases, its resynthesis is mainly mediated by mitochondrial F0F1 ATP synthase, which couples the transport of protons across the mitochondrial inner membrane to the synthesis of ATP (6, 7, 12). However, when the proton electrochemical gradient across the inner membrane collapses, the enzyme switches its catalytic activity from ATP synthesis to ATP hydrolysis (12). Although the hydrolytic activity during ischemia counteracts the ischemia-induced collapse of the proton electrochemical gradient across the inner membrane, F0F1 ATP synthase is a major consumer of ATP under ischemic conditions (12, 37). The modulation of F0F1 ATP synthase hydrolytic activity during ischemia may therefore play an important role in maintaining the intracellular ATP levels (2, 40, 48). The inhibition of F0F1 ATP synthase hydrolytic activity during ischemia is mediated by the interaction between the enzyme and the inhibitor protein IF1, which modulates the enzyme activity by reversibly binding to one of the three {beta}-subunits of the catalytic sector F1 (10, 15, 25). Hence, IF1 binding/release to/from the F0F1 ATP synthase complex may be a physiological regulator of the enzyme activity (12).

Ischemic preconditioning (IP), which is achieved by brief (a few minutes) episodes of coronary occlusion, is well known to induce myocardial protection against the injury caused by sustained ischemia followed by reperfusion (27). Among other effects, IP induces a sort of "metabolic hibernation," characterized by a reduction in the ATP pool combined with a reduction in ATP hydrolytic rate during subsequent ischemic stress (17, 19, 28, 35, 43). The duration (minutes or days) of the reduction in the ATP pool depends on the duration of the preconditioning ischemia, whereas the reduction in ATP hydrolysis is consistent with a reduction in metabolic demand (17, 19, 28, 35, 43).

Coronary reactive hyperemia (CRH) is the increase in flow that follows a coronary occlusion brought about primarily by the release of vasoactive compounds from ischemic myocardium, with the contribution of a myogenic mechanism and endothelial factors (9, 13, 46). For instance, CRH can be obtained with 15 s of coronary occlusion, which has been demonstrated not to cause preconditioning and not to alter the hyperemic response, even if repeated several times at intervals of only 4 min (32). It has been shown that when IP is accomplished in dogs and goats with 5–10 min of ischemia, the magnitude and duration of a CRH subsequent to the 15-s occlusion are reduced (8, 20, 30, 32).

Several studies (2, 7, 48) have suggested that IF1 could mediate the inhibition of F0F1 ATP synthase observed in preconditioned myocardium. However, contradictory results have been reported (11, 47). In particular, data are lacking on the relation between the ATP-to-ADP ratio (ATP/ADP) and F0F1 ATP synthase activity during a CRH elicited by a few seconds of occlusion in normal and preconditioned hearts.

Because myocardial metabolism is strictly aerobic, we hypothesize that F0F1 ATP synthase may be modulated by the inhibitor protein IF1 during a CRH, even if CRH is elicited by only 15 s of ischemia. Moreover, because CRH is blunted by IP, we hypothesize that F0F1 ATP synthase may be inhibited in such a condition. Hence, the goals of this study are 1) to assess the dynamics of F0F1 ATP synthase activity, ATP concentration, and ATP/ADP during and after CRH, 2) to determine whether IP affects these dynamics, and 3) to determine whether the reduction of CRH after IP (8, 30, 32) is accompanied by variations in F0F1 ATP synthase activity. To accomplish these goals, we assayed ATP concentration, ATP/ADP, and F0F1 ATP synthase activity in myocardial biopsies obtained before, during, and after CRH elicited by 15 s of ischemia, with and without IP.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (DL 116, 27 January 1992, published in the Gazzetta Ufficiale della Repubblica Italiana, no. 40, 18 February 1992) and conformed to the Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). The experiments were approved by the Ethical Committee of the University of Turin.

Experimental model. The experiments were carried out in 10 anesthetized goats weighing 45–60 kg. Anesthesia was induced by injection of ketamine hydrochloride (15 mg/kg iv) and was maintained by ventilation with 2:1 nitrous oxide-oxygen and a continuous infusion of ketamine hydrochloride (24 mg·kg–1·h–1). Fentanyl (Fentanest, Pharmacia; Milan, Italy) was injected hourly (1 mg/kg) (8, 30, 32). The right external jugular vein and both common carotid arteries were isolated through a ventral incision in the neck. A catheter was inserted into the jugular vein for administration of solutions needed to maintain anesthesia and acid-base balance within normal limits. Catheters were inserted into the two common carotid arteries. One of the catheters, connected to an electromanometer, was pushed into the aortic root to record aortic blood pressure (ABP); the other was used to obtain samples of arterial blood for the assessment of acid-base balance and respiratory conditions of the animals. Needle electrodes were inserted into the muscles of the proximal part of the limbs to record ECG from one of the limb leads.

The chest was opened by left lateral thoracotomy, and the heart was suspended in a pericardial cradle. The proximal part of the left circumflex coronary artery (LCCA) was gently isolated, and a flow probe, connected to an electromagnetic flowmeter, was placed around the LCCA to record coronary blood flow (CBF). Distal to this flow probe, a snare was placed around the artery. The snare was used to occlude the artery to obtain zero flow level and to produce CRH and IP. The temperature was monitored by means of an esophageal thermistor probe (model DU-3, Ellab; Copenhagen, Denmark) and was maintained within normal limits using an electric heating pad and/or an infrared lamp. The respiration and acid-base status of animals were monitored using a gas analyzer (model 280, Ciba-Corning; Halstead, Essex, UK). PO2 and PCO2 were kept within the normal limits of the goat (24) by regulation of pump rate and tidal volume and/or addition of oxygen to the inspired air.

ABP, CBF, and ECG were recorded with a cassette data recorder (model R-71, TEAC, Tokyo, Japan) and reproduced using an electrostatic paper recorder (model ES-1000, Gould, Paris, France). The mean ABP was obtained by graphic integration of the relevant curve. CBF was measured in diastole, when myocardial contraction does not affect coronary resistance. Resting diastolic CBF and the following variables of CRH were determined: diastolic CBF at the peak of the hyperemia, duration of the hyperemia, and total hyperemic flow. The duration of the hyperemic response was calculated as the time between the release of the occlusion and the instant the flow had returned to ≤5% from the control value. Total hyperemic flow was defined as the area between the flow trace and the zero line over the duration of the response.

Experimental protocol. Eight animals underwent the protocol described in Fig. 1. Briefly, before preconditioning, baseline data were recorded as nonpreconditioned control (Cnp), when a steady-state condition was attained. This typically occurred within 30 min after the preparation was completed. Then, a first CRH was induced (CRHnp). After collection of baseline data, IP was induced. After preconditioning, again control data (Cprec) were recorded and a second CRH was induced (CRHprec).



View larger version (10K):
[in this window]
[in a new window]
  		Fig. 1. Experimental protocol. Downward arrows, time at which biopsies were obtained in the 8 animals; dashed lines, schematic representation of coronary blood flow (CBF) during coronary reactive hyperemia (CRH). Time lines are related to onset of hyperemias. Cnp and Cprec, nonpreconditioned and preconditioned controls, respectively; CRHnp and CRHprec, nonpreconditioned and preconditioned CRH, respectively.

 
To induce CRH, the LCCA was occluded with the snare distal to the flow probe and released after 15 s. To induce IP, the LCCA was occluded twice for 2.5 min each, with an interval of 5 min between occlusions. Such a combination of ischemia and reperfusion falls within the temporal limits required to induce myocardial and vascular preconditioning in all investigated animal species (31, 43). The second CRH (CRHprec) was induced as described for CRHnp at ~10 min after IP, i.e., after the flow was normalized and Cprec data were collected.

In two additional animals, CRHprec was not induced to assess the effects of IP alone.

Biopsies. In eight animals, seven cardiac biopsies (20–30 mg) were obtained as described by Scholz and Balaban (42) from the area perfused by the LCCA. Biopsies were obtained in the control before IP (Cnp), during hyperemia (20 s), and 4 and 6 min after the beginning of CRHnp, as well as in the control after IP (Cprec, i.e., 10 min after release of the second 2.5-min occlusion of the IP protocol), during hyperemia (20 s), and 4 min after the beginning of CRHprec. In four of these eight animals, biopsies were also obtained 30 min after Cprec. In the two additional experiments, biopsies were obtained 10 min after IP (i.e., Cprec), as well as 4 and 30 min after Cprec. In all animals, biopsies were also obtained at the end of the experiments from an area distal to the risk area (remote control). Biopsies were rapidly divided into two parts: one was immediately used to measure F0F1 ATP synthase activity, and the other was immediately frozen in liquid nitrogen and stored at –80°C until it was used for nucleotide assay or electrophoretic analysis (see below). The area perfused by the LCCA was visually identified on the basis of the distribution of epicardial branches of the artery. At the end of the experiments, the vascular bed in which the sites of the biopsies were included was confirmed by occluding the LCCA and perfusing the heart with monastral blue.

Analytic procedures. F0F1 ATP synthase activity was always measured as the maximal ATP hydrolytic activity (Vmax) and is reported as micromoles of ATP per minute per milligram of protein (U/mg). The fresh parts of the biopsies were used. In the case of Cnp, the sample was divided into three pieces; in the other cases, the samples were divided into two pieces.

The first piece of all biopsies (~3 mg of tissue) was homogenized by brief (3 times for 20 s each) sonication (model UP 50H dr, Hielscher; Berlin, Germany) to expose the F1 sector to the solvent. To avoid release of IF1 from the enzyme, sonication was performed under controlled conditions in 0.25 ml of ice-cold low-salt buffer (20 mM HEPES, 1 mM MgCl2, and 2 mM EGTA, pH 7.2). This buffer was low in Ca2+ and Na+ to limit any contribution from ATPases other than F0F1 ATP synthase. The Vmax of F0F1 ATP synthase was then measured as described by Comelli et al. (4). In all samples, >90% of the ATP hydrolytic activity assayed was oligomycin sensitive and <6% was sensitive to vanadate or ouabain, demonstrating that the activity was mainly due to mitochondrial F0F1 ATP synthase. The activity was assayed at 37°C in the presence of an ATP-regenerating system by coupling ATP hydrolysis to NADH oxidation (37) and was monitored spectrophotometrically at a wavelength of 340 nm.

A partial or maximal increase in enzyme activity can be achieved in vitro when the IF1-F0F1 ATP synthase complex is exposed to high ionic concentration, which induces a partial release of IF1, or to high pH, which induces an almost complete release of IF1 (34, 38, 40, 42). Then, to provide an indication of IF1 involvement in the enzyme modulation in vivo, the second piece of the fresh biopsy was sonicated in the presence of 150 mM KCl, which is known to induce the partial IF1 release (42).

Finally, the sonication of the third piece of the Cnp biopsy was performed in the presence of 0.25 M sucrose, 1 mM ATP, and 2 mM EDTA, with pH adjusted to 9.2 with NH4OH. This procedure stripped away IF1 (40) and allowed us to estimate the potential maximal activity elicited by the enzyme. Protein concentration was determined by the method described by Lowry et al. (22).

To perform the electrophoretic analysis, frozen biopsies (10–15 mg) were treated with n-dodecylmaltoside in aminocaproic acid, and the extracts were subjected to blue native electrophoresis (BN-PAGE) (45). Then, the gels were stained with Coomassie blue G, and the banding patterns were scanned using a LKB Ultrascan XL densitometer. The protein concentration of the detergent extracts was determined by the method described by Bradford (3).

Part of the frozen tissue was extracted with 0.5 M perchloric acid, neutralized, and analyzed for ATP and ADP by high-pressure liquid chromatography as described elsewhere (26).

Statistics. Values are means ± SE. For statistical analysis, one-way ANOVA with Tukey's honestly significant difference post hoc test was used to evaluate separately the statistical significance of the differences of the parameter before and after IP (i.e., from Cnp to 6 min after CRHnp and from Cprec to 4 min after CRHprec). Student's t-test for paired data (2-tailed) was used to evaluate the statistical significance of the differences between Cnp and Cprec and the difference of total hyperemic flow and duration of CRH before and after IP. In all cases, P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hemodynamic data. Figure 2 shows traces of ABP and CBF with CRH before and after IP. No significant changes were observed for ABP and heart rate before and after IP. IP did not affect basal CBF (59 ± 3 vs. 58 ± 3 ml/min) but induced remarkable changes in CRH. The relevant data are reported in Table 1. As expected (8, 20, 30, 32), IP caused a 23% reduction (from 248 ± 8 to 190 ± 7 ml/min, P < 0.01, Student's t-test) in total hyperemic flow. This reduction was associated with a 12% reduction (from 150 ± 8 to 132 ± 5 s, P < 0.01, Student's t-test) of CRH duration.



View larger version (70K):
[in this window]
[in a new window]
  		Fig. 2. Aortic blood pressure (ABP) and CBF during CRH before (A) and after (B) ischemic preconditioning.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Hemodynamic and biochemical variables before and after preconditioning

 
Biochemical data. Inasmuch as the biopsies were rather small in two animals, not all experiments were successful. Therefore, in eight animals (6 of the group of 8 animals + 2 additional animals), all the pieces of biopsies (see Analytic procedures) were used to determine all studied parameters.

Effect of CRH on F0F1 ATP synthase activity before and after IP. Vmax of F0F1 ATP synthase was assayed at the times indicated by downward arrows in Fig. 1. Data are reported in Table 1. F0F1 ATP synthase activity significantly (P < 0.05) increased from 0.99 ± 0.03 U/mg in Cnp to 1.20 ± 0.10 U/mg during CRHnp. At 4 min after the beginning of CRHnp, when blood flow returned to the level at Cnp, F0F1 ATP synthase activity was low (0.72 ± 0.035 U/mg, P < 0.05 vs. Cnp) and then recovered to near baseline [0.97 ± 0.05 U/mg, P = not significant (NS) vs. Cnp] 2 min later, i.e., 6 min after the beginning of CRHnp.

At 10 min after IP (Cprec), F0F1 ATP synthase activity was 0.87 ± 0.04 U/mg, which is significantly (P < 0.05, Student's t-test) less than in Cnp. During CRHprec, F0F1 ATP synthase activity was 1.00 ± 0.03 U/mg, which is not significantly different from that recorded in Cprec. At 4 min after the beginning of CRHprec, F0F1 ATP synthase activity showed almost the same value (0.88 ± 0.06 U/mg) as in Cprec. In the animals in which we assessed the effects of IP, F0F1 ATP synthase activity remained as low as at Cprec until 30 min after IP (0.90 ± 0.02 U/mg, P = NS vs. Cprec).

Modulation of F0F1 ATP synthase during CRH before and after IP. The transient changes in enzyme activity suggested that F0F1 ATP synthase is reversibly modulated as a consequence of the different experimental maneuvers. The putative candidate for this modulation is IF1. To test this hypothesis, we measured the response of F0F1 ATP synthase hydrolytic activity assayed after each experimental maneuver to a high-salt buffer (150 mM KCl), which is known to increase the enzyme activity as a consequence of partial IF1 release (42). The activation percentages calculated for each time point are reported in Table 1.

In the biopsies of nonpreconditioned heart, the activation percentage induced by high-salt buffer fell from 20% in Cnp to 3% during CRHnp and increased to 45% 4 min after the beginning of CRHnp (P < 0.05 vs. Cnp for both). It returned to the Cnp level 6 min after the artery was reopened. After IP, however, the percentage increased to 31% in Cprec (P < 0.05 vs. Cnp, Student's t-test); then it was 19% (P = NS vs. Cprec) during CRHprec and 25% (P = NS vs. Cprec) 4 min after the beginning of CRHprec. These data indicate that, before KCl treatment, F0F1 ATP synthase was inhibited 4 min after the beginning of CRHnp, as well as 10 min after IP in Cprec, in contrast to our observation during CRHnp, when F0F1 ATP synthase inhibition by IF1 was low.

Because the sonication of the biopsies in high-pH buffer (pH 9.2) stripped away ≥95% of the endogenous IF1 from F0F1 ATP synthase (40), we consider the ratio of the enzyme activity measured after sonication under routine conditions in low-salt buffer at pH 7.2 to that measured in control biopsies after sonication at pH 9.2 to be an index of the amount of the enzyme inhibited by IF1 (Table 1). Specifically, the amount of inhibited enzyme decreased significantly from 63 ± 2% in Cnp to 54 ± 3% in CRHnp (P < 0.05). At 4 min after the beginning of CRHnp, the percentage increased to 72 ± 2% (P < 0.05 vs. Cnp) and returned to Cnp 6 min after the artery was reopened (63 ± 2%, P = NS vs. Cnp). The percentage of inactive enzyme (67 ± 2%) was significantly higher in Cprec than in Cnp (P < 0.05, Student's t-test), indicating that IP favored the binding of IF1 to F0F1 ATP synthase. Finally, Tukey's honestly significant difference post hoc tests revealed that the changes during and after CRHprec were not significant with respect to Cprec.

Although the percentages of the enzyme inhibited by IF1 change significantly in response to CRHnp and IP, they always remain high. This is a well-known phenomenon, the meaning of which is debated (6, 7, 12, 38).

To exclude alterations in the synthesis and/or proteolytic degradation of the enzyme, we analyzed the detergent extracts of biopsies with BN-PAGE. The densitometric analysis of gels did not reveal, in the various samples, any significant change in the content of F0F1 ATP synthase or mitochondrial complexes (Fig. 3), indicating that the changes in activity were not due to alterations in enzyme content.



View larger version (43K):
[in this window]
[in a new window]
  		Fig. 3. Blue-native electrophoresis (BN-PAGE) of mitochondrial oxidative phosphorylation complexes in biopsies of goat heart during CRH before and after ischemic preconditioning. A: myocardial biopsies obtained from baseline were treated with n-dodecylmaltoside, and oxidative phosphorylation complexes were separated by BN-PAGE and stained with Coomassie blue G. Different quantities of proteins (8–19 µg) were loaded onto the gel. Coomassie-stained band areas of complex V obtained from densitometric analysis of different gels were correlated to protein quantities loaded. B: biopsies were collected during experimental maneuvers, frozen in liquid N2, and stored at –80°C until use. Detergent extracts from myocardial specimens and bovine heart mitochondria were run on BN-PAGE and stained with Coomassie blue G. Bovine heart mitochondria were used as standard for identification of oxidative phosphorylation complexes as described elsewhere (21). Lanes are loaded with 15 µg of extract proteins in the case of goat heart biopsies or with 20 µg of extract proteins in the case of bovine heart mitochondria. Lane 1, Cnp; lane 2, CRHnp; lane 3, 4 min after beginning of CRHnp; lane 4, 6 min after beginning of CRHnp; lane 5, Cprec; lane 6, CRHprec; lane 7, 4 min after beginning of CRHprec; lane 8, bovine heart mitochondria. C: band area of complex V was detected by densitometric analysis of different gels and reported as arbitrary units (P = NS). Percentage of complex V was calculated with respect to total area of complexes I, V, and III taken as 100%.

 
ATP concentration and ATP/ADP. Data for ATP concentration and ATP/ADP are reported in Table 1. During Cnp, ATP concentration was 3.74 ± 0.6 µmol/g, and ATP/ADP was 7.38 ± 0.45. ATP concentration fell to 2.52 ± 0.82 µmol/g (P = NS vs. Cnp) and 1.77 ± 0.44 µmol/g (P < 0.05 vs. Cnp) during CRHnp and 4 min after the beginning of CRHnp, respectively. ATP/ADP was reduced by about one-half during CRHnp (3.95 ± 0.61, P = 0.01 vs. Cnp) and 4 min after the beginning of CRHnp (3.43 ± 0.51, P = 0.01 vs. Cnp). At 6 min, ATP concentration and ATP/ADP began to rise again to 2.80 ± 0.68 µmol/g and 5.24 ± 0.61, respectively (P = NS vs. Cnp for both). After IP, in Cprec, ATP concentration and ATP/ADP were 1.58 ± 0.48 µmol/g and 4.28 ± 0.65, respectively. Both variables were significantly (P < 0.001, Student's t-test) reduced with respect to Cnp. In CRHprec, ATP concentration and ATP/ADP rose sharply to 3.47 ± 079 µmol/g (P < 0.05 vs. Cprec) and 10.43 ± 2.43 (P = NS), respectively. The two variables were 2.49 ± 0.63 µmol/g and 15.30 ± 5.26, respectively, 4 min after the beginning of CRHprec, both showing a significant (P < 0.05 vs. Cprec) increase. Despite an overall increase, the change in ATP/ADP during CRHprec was not significant because of data dispersion.

In the experiments where we assessed the effects of IP, a sharp increase in ATP/ADP (10.13 ± 2.67) was also observed 4 min after Cprec. This increase was very similar to that observed in the other experiments 4 min after the beginning of CRHprec. Finally, 30 min after Cprec, the ratio returned to Cnp.

The values of all biochemical variables obtained from remote controls were not significantly different from those obtained in Cnp, showing no time-dependent deterioration of the heart throughout the experiment.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study indicates that brief (15 s) periods of ischemia are able to induce transient variations of F0F1 ATP synthase activity and the ATP pool before, but not after, preconditioning, when the enzyme activity is blunted for a long time. Our data strongly indicate that the F0F1 ATP synthase activity modulation during CRH (up- and downregulation) and after IP is due to the release/binding of IF1 to the enzyme. Moreover, this study confirms that there is little relation between F0F1 ATP synthase activity and the ATP pool in preconditioned hearts. In fact, the reduction in F0F1 ATP synthase activity was associated with a reduction in tissue ATP concentration and ATP/ADP during Cprec, whereas during and after (4 min and 30 min) CRHprec, the two parameters did not appear to be related, perhaps because F0F1 ATP synthase and cytoplasmic ATPase activities are reduced in preconditioned hearts (2, 17, 19, 33, 36, 44, 48). Therefore, the ATP pool is the result of the relative activity of these two kinds of enzymes.

Hemodynamic measurements. In the present study, as well as in previous studies (8, 30, 32), IP does not alter resting heart rate, ABP, and CBF. In contrast, total hyperemic flow during CRHprec was markedly reduced. In a previous investigation, the reduction of the total flow of reactive hyperemia after IP was attributed to a reduced myocardial oxygen demand (8). The fact that the amplitude of total hyperemic flow depends on the changes in postischemic myocardial metabolism induced by IP was further supported by studies where mitochondrial ATP-sensitive K+ channels were activated with diazoxide (32). The observation that heart rate and blood pressure were not affected by IP suggests that the reduction in oxygen demand in preconditioned hearts does not reflect any remarkable impairment of the external cardiac performance.

F0F1 ATP synthase activity during CRHnp. Our results suggest that F0F1 ATP synthase was modulated by IF1 during and after CRHnp.

With respect to the increased activity of F0F1 ATP synthase observed during CRHnp, several hypotheses can be entertained. The fundamental mechanism triggering CRH is mediated primarily by the production of local metabolic vasodilators released from cardiomyocytes secondary to the oxygen delivery-demand mismatch during the preceding coronary occlusion (8, 9, 13, 23, 46). In such a condition, ATP hydrolysis prevails over ATP synthesis, although the ischemia lasted only 15 s. Therefore, the reduction of myocardial ATP concentration caused by the hydrolysis may have contributed to the increased F0F1 ATP synthase activity through a feedback mechanism mediated by IF1 release (21). Moreover, the increase in enzyme activity can be attributed to enhanced myocardial oxygen delivery, associated with increased coronary flow when a reduction of ATP concentration is likely to trigger ATP resynthesis (14). This is consistent with the finding that F0F1 ATP synthase increases in response to physiological increases in myocardial work induced by infusion of phenylephrine in the dog (42).

After CRHnp, F0F1 ATP synthase activity returned to baseline 6 min after release of the artery. The transient reduction of the activity observed 4 min after the beginning of CRHnp is intriguing. Although it is mediated by IF1 binding, it is difficult to explain. We can argue that the increase of enzyme activity during CRHnp, followed by a reduction a few minutes later, is somehow correlated to the initial increase and the subsequent decrease in oxygen uptake that occur during reactive hyperemia (41). Moreover, we hypothesize that this transient inhibition may be interpreted as a counterpart of the long-lasting inhibition observed after longer/preconditioning ischemia (present study; 2, 7, 48; see below).

F0F1 ATP synthase activity after IP. After IP, F0F1 ATP synthase activity was low for ≥30 min. Such an inhibition was due to higher IF1 binding to the enzyme with respect to Cnp, as suggested by the activation observed with KCl treatment of biopsies (31% vs. 20%; Table 1). In such a condition of long-lasting inhibition of the enzyme, CRHprec was not able to change significantly F0F1 ATP synthase activity with respect to Cprec, in contrast to CRHnp with respect to Cnp. The fact that, during the 15-s occlusion leading to CRHprec, ATP hydrolysis occurred to a lesser extent (35, 36) than during the occlusion leading to CRHnp, thereby not inducing the feedback activation mechanism mediated by IF1 release, may have contributed to this relative unresponsiveness of the enzyme. This hypothesis is consistent with the relation between the reduction of CRH after IP and the decreased activity of F0F1 ATP synthase. In other words, because the hydrolytic activity of the enzymes (F0F1 ATP synthase and nonmitochondrial ATPases) is blunted during the preceding coronary occlusion, it is likely that the production of local metabolic vasodilators (e.g., adenosine) from cardiomyocytes is reduced.

Because ATP concentration is low in Cprec, the binding of IF1 may be triggered by the metabolic alterations (i.e., high lactate and creatine phosphate/phosphate overshoot) observed after preconditioning ischemia (10, 18, 33). The subsequent increase in ATP concentration may be responsible for the sustained inhibition of the enzyme (see below). Mitochondrial K+ channel opening, which is a pivotal step of IP, may also be involved in IF1 binding to the enzyme, as recently suggested from in vitro studies (1, 5).

ATP concentration and ATP/ADP. No apparent correlation was found between F0F1 ATP synthase activity and ATP concentration and ATP/ADP. Because ATP concentration and ATP/ADP and, hence, the cell energy state depend essentially on the cell energy demand-supply equilibrium, it is not surprising that the two factors are not strictly linked during acute perturbations of coronary flow. In other words, ATP concentration reflects the overall activity of F0F1 ATP synthase and ATPases until the sampling time, rather than the activity of the enzymes at that time.

ATP concentration and ATP/ADP during CRHnp. It is likely that, during the occlusion leading to CRHnp, ATP concentration and ATP/ADP decreased as a consequence of prevailing ATP hydrolysis due to the lack of oxygen during the 15 s of occlusion. It is then possible that, during CRHnp, ATP concentration did not have enough time to recover fully, despite the transient activation of F0F1 ATP synthase above the control value. Moreover, the reduction of the enzyme activity 4 min after the beginning of CRHnp may have contributed to the decrease of ATP concentration at this time and to the incomplete recovery of ATP/ADP 6 min after release of the artery when F0F1 ATP synthase activity returns to the control level.

ATP concentration and ATP/ADP after IP. IP is known to depress cell ATP concentration (17, 19, 35, 36, 44). In particular, Jennings et al. (17) reported that ischemic preconditioning in the dog (10 min of ischemia + 10 min of reperfusion) reduces ATP by 30% and slightly reduces ADP (–25%). Therefore, because of the prevalence of reduction in ATP concentration, ATP/ADP is also reduced after IP. It is then likely that the decrease in ATP concentration and ATP/ADP observed after IP (i.e., in Cprec) is the consequence of the total of 5 min of ischemia of IP (2 periods of 2.5 min each of ischemia), during which ATP hydrolysis prevails over ATP resynthesis. In our model, ATP concentration and ATP/ADP decreased by a larger extent than observed by Jennings et al. (16, 17) in the dog, probably due to species difference, which implies different degrees of inhibition of mitochondrial ATPase activity (10).

The significant increase in ATP concentration and ATP/ADP 4 min after the beginning of CRHprec as well as 4 min after Cprec, when CRH was not induced (the 2 additional experiments), might be due to the reduced rate of ATP hydrolysis by cytoplasmic ATPases, as documented in preconditioned hearts (17, 19, 33, 35, 44). The enhanced exogenous glucose uptake and glycolysis that occur in preconditioned heart might also contribute to the increase in ATP concentration (29, 44). Both of these mechanisms may be associated with the partial recovery of mitochondrial oxidative phosphorylation after preconditioning ischemia (17, 29). Indeed, when measured 30 min after Cprec, ATP/ADP returns to the same value observed in Cnp, indicating that the sharp increase in CRHprec and 4 min after Cprec might be attributed to a transient decrease in oxygen demand characterizing early preconditioning. These findings are in agreement with the fact that 2–3 min of ischemia are followed by recovery of the adenylate pool in minutes or hours, rather than in days, as required by longer periods of ischemia (19).

In summary, after preconditioning ischemia, the ATP pool continues to increase, while ATP synthesis by F0F1 ATP synthase is reduced. It is then likely that the increase in the ATP pool is sustained by inhibition of cytoplasmic ATPases. On the other hand, the increasing ATP pool contributes to the blunting of F0F1 ATP synthase activity during early preconditioning. Therefore, perturbations, such as 15 s of occlusion and CRH, are not able to significantly change the activity of F0F1 ATP synthase. Also, the pattern of variations of F0F1 ATP synthase activity and ATP/ADP we observed is consistent with the reduced hyperemic flow and with the reported reduction of ATP hydrolysis when a coronary occlusion is performed in preconditioned hearts (17, 19, 28, 35, 43, 44).

Limits of the study. One of the limits of this procedure concerns the ability to perform biopsies that would reduce to a minimum the time required for freeze clamping of the tissue before significant changes occur in ATP/ADP. This time was, in most cases, kept to <2 s, thereby allowing minimal changes in ATP/ADP. However, to limit myocardial injury, the size of the samples was rather small. Despite optimization in the extraction procedure, the small size of the samples often induced an unfavorable signal-to-noise ratio, which precluded analysis and quantification of chromatographic peaks lower than those of ADP and ATP, namely, all the other metabolites of ATP (AMP, adenosine, inosine, hypoxanthine, and xanthine). Moreover, elution of creatine and creatine phosphate, which normally occurs early in the chromatogram together with inosine, hypoxanthine, and xanthine, was difficult to discern for quantitative purposes.

It must be also stressed that an appropriate control group is missing in this study. Such a group would have required a larger number of animals. Although this flaw may weaken our conclusions with regard to the effects of the experimental maneuvers on the observed changes, the fact that remote control data are similar to Cnp data, as well as the consistency of the results obtained in the two additional animals, suggests that the changes described in this study depend on the maneuvers, rather than on the elapsed time. Inasmuch as the purpose of this study was to focus on the effects of the single maneuver, we used as reference points the values measured before each individual maneuver.

In conclusion, very short (a few seconds) episodes of ischemia induce transient variations of F0F1 ATP synthase activity, which may explain why ischemias of such a duration are unable to induce preconditioning. The activity of F0F1 ATP synthase transiently increases during CRHnp and transiently decreases immediately after the hyperemia, but after 6 min it returns to control. Whether these variations can be attributed to a feedback mechanism triggered by low ATP concentration during coronary occlusion and/or to oxygen uptake variations during CRH remains to be elucidated.

On the contrary, longer (minutes) periods of ischemia are able to blunt F0F1 ATP synthase activity for a longer time, and subsequent periods of ischemia are not able to up- or downregulate this enzyme further. After IP, F0F1 ATP synthase activity is blunted during Cprec, and it does not increase significantly during CRHprec. This inhibition is not reversed 30 min after Cprec. This suggests that such inhibition is slowly reversible and may play an important role in the preservation of ATP during prolonged ischemia, when F0F1 ATP synthase becomes a major ATP consumer.

We provide evidence that, under all conditions before and after IP, inhibition of F0F1 ATP synthase activity is due to enzyme modulation by IF1 binding.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Compagnia di San Paolo, Torino, the Consorzio Interuniversitario per la Ricerca Cardiovascolare, the Programma Operativo Regione Friuli-Venezia Giulia (Misura D4), and the Italian Ministry of Education, University, and Research (MIUR-Cofin 2002).


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Pagliaro, Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Ospedale S. Luigi, Regione Gonzole, 10043 Orbassano (TO), Italy (E-mail: pasquale.pagliaro{at}unito.it)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ala-RämiA, Ylitalo KV, and Hassinen IE. Ischaemic preconditioning and a mitochondrial KATP channel opener both produce cardioprotection accompanied by F1F0-ATPase inhibition in early ischaemia. Basic Res Cardiol 98: 1–9, 2003.[CrossRef][Web of Science][Medline]
  2. BosettiF, Yu G, Zucchi R, Ronca-Testoni S, and Solaini G. Myocardial ischemic preconditioning and mitochondrial F0F1-ATPase activity. Mol Cell Biochem 215: 31–37, 2000.[CrossRef][Web of Science][Medline]
  3. BradfordMM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 74: 248–254, 1976.[CrossRef]
  4. ComelliM, Di Pancrazio F, and Mavelli I. Apoptosis is induced by decline of mitochondrial ATP synthesis in erythroleukemia cells. Free Radic Biol Med 34: 1190–1199, 2003.[CrossRef][Web of Science][Medline]
  5. ContessiS, Metelli G, Mavelli I, and Lippe G. Diazoxide affects the IF1 inhibitor protein binding to F1 sector of beef heart F0F1-ATP synthase. Biochem Pharmacol 67: 1843–1851, 2004.[CrossRef][Web of Science][Medline]
  6. DasAM and Harris DA. Control of mitochondrial ATP synthase in heart cells: inactive to active transitions caused by beating or positive inotropic agents. Cardiovasc Res 24: 411–417, 1990.[Abstract/Free Full Text]
  7. DasAM and Harris DA. Regulation of the mitochondrial ATP synthase in intact rat cardiomyocytes. Biochem J 266: 355–361, 1990.[Web of Science][Medline]
  8. GattulloD, Linden RJ, Losano G, Pagliaro P, and Westerhof N. Ischaemic preconditioning changes the pattern of coronary reactive hyperaemia in the goat: role of adenosine and nitric oxide. Cardiovasc Res 42: 57–64, 1999.[Abstract/Free Full Text]
  9. GattulloD, Pagliaro P, Linden RJ, Merletti A, and Losano G. The role of nitric oxide in the initiation and in the duration of some vasodilator responses in the coronary circulation. Pflügers Arch 430: 96–104, 1995.[CrossRef][Web of Science][Medline]
  10. GreenDW and Grover GJ. The IF1 inhibitor protein of the mitochondrial F1F0-ATPase. Biochim Biophys Acta 1458: 343–355, 2000.[Medline]
  11. GreenDW, Murray HN, Sleph PG, Wang FL, Baird AJ, Rogers WL, and Grover GJ. Preconditioning in rat hearts is independent of mitochondrial F1F0 ATPase inhibition. Am J Physiol Heart Circ Physiol 274: H90–H97, 1998.[Abstract/Free Full Text]
  12. HarrisDA and Das AM. Control of mitochondrial ATP synthesis in the heart. Biochem J 280: 561–573, 1991.
  13. IshibashiY, Duncker DJ, Zhang J, and Bache RJ. ATP-sensitive K+ channels, adenosine, and nitric oxide-mediated mechanisms account for coronary vasodilation during exercise. Circ Res 82: 346–359, 1998.[Abstract/Free Full Text]
  14. IwamotoT, Bai XJ, and Downey HF. Coronary perfusion-related changes in myocardial contractile force and systolic ventricular stiffness. Cardiovasc Res 28: 1331–1336, 1994.[Abstract/Free Full Text]
  15. JacksonPJ and Harris DA. The mitochondrial ATP synthase inhibitor protein binds near the C-terminus of the F1 {beta}-subunit. FEBS Lett 229: 224–228, 1988.[CrossRef][Web of Science][Medline]
  16. JenningsRB, Schaper J, Hill ML, Steenbergen C Jr, and Reimer KA. Effect of reperfusion late in the phase of reversible ischemic injury. Changes in cell volume, electrolytes, metabolites, and ultrastructure. Circ Res 56: 262–278, 1985.[Abstract/Free Full Text]
  17. JenningsRB, Sebbag L, Schwartz LM, Crago MS, and Reimer KA. Metabolism of preconditioned myocardium: effect of loss and reinstatement of cardioprotection. J Mol Cell Cardiol 33: 1571–1588, 2001.[CrossRef][Web of Science][Medline]
  18. KleinG, Satre M, Dianoux AC, and Vignais PV. Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase. Biochim Biophys Acta 636: 185–192, 1981.[Medline]
  19. KlonerRA and Jennings RB. Consequences of brief ischemia: stunning, preconditioning, and their clinical implications. Part 1. Circulation 104: 2981–2989, 2001.[Abstract/Free Full Text]
  20. LaxsonDD, Homans DC, Dai XZ, Sublett E, and Bache RJ. Oxygen consumption and coronary reactivity in postischemic myocardium. Circ Res 64: 9–20, 1989.[Abstract/Free Full Text]
  21. LippeG, Sorgato MC, and Harris DA. The binding and release of the inhibitor protein are governed independently by ATP and membrane potential in ox-heart submitochondrial vesicles. Biochim Biophys Acta 933: 12–21, 1988.[Medline]
  22. LowryOH, Rosenbrough NJ, Farr AL, and Randall RJ. Protein measurement with Folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  23. MalletRT, Shang-Chiun L, and Downey HF. Endogenous adenosine increases O2 utilisation efficiency in isoprenaline-stimulated canine myocardium. Cardiovasc Res 31: 102–116, 1996.[CrossRef][Web of Science][Medline]
  24. McCutcheonFH. Respiration and circulation. In: Biology Data Book, edited by Altman PL and Dittmer DS. Washington, DC: FASEB, 1964, p. 219–243.
  25. MilgromYM. When beef-heart mitochondrial F1-ATPase is inhibited by inhibitor protein a nucleotide is trapped in one of the catalytic sites. Eur J Biochem 200: 789–795, 1991.[Web of Science][Medline]
  26. MotterliniR, Samaja M, Tarantola M, Micheletti R, and Bianchi G. Functional and metabolic effects of propionyl-L-carnitine in the isolated perfused hypertrophied rat heart. Mol Cell Biochem 116: 139–145, 1992.[CrossRef][Web of Science][Medline]
  27. MurryCE, Jennings RB, and Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 74: 1124–1136, 1986.[Abstract/Free Full Text]
  28. MurryCE, Richard VJ, Reimer KA, and Jennings RB. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ Res 66: 913–931, 1990.[Abstract/Free Full Text]
  29. OpieLH and Sack MN. Metabolic plasticity and the promotion of cardiac protection in ischemia and ischemic preconditioning. J Mol Cell Cardiol 34: 1077–1089, 2002.[CrossRef][Web of Science][Medline]
  30. PagliaroP, Chiribiri A, Rastaldo R, Mancardi D, Penna C, Gattullo D, and Losano G. Ischemic preconditioning changes the pattern of coronary reactive hyperemia regardless of mitochondrial ATP-sensitive K+ channel blockade. Life Sci 71: 2299–2309, 2002.[CrossRef][Web of Science][Medline]
  31. PagliaroP, Gattullo D, Rastaldo R, and Losano G. Ischemic preconditioning: from the first to the second window of protection. Life Sci 69: 1–15, 2001.[CrossRef][Web of Science][Medline]
  32. PagliaroP, Rastaldo R, Losano G, and Gattullo D. Mitochondrial ATP-sensitive channel opener does not induce vascular preconditioning, but potentiates the effect of a preconditioning ischemia on coronary reactive hyperemia in the anesthetized goat. Pflügers Arch 443: 166–174, 2001.[CrossRef][Web of Science][Medline]
  33. PucarD, Dzeja PP, Bast P, Juranic N, Macura S, and Terzic A. Cellular energetics in the preconditioned state. J Biol Chem 276: 44812–44819, 2001.[Abstract/Free Full Text]
  34. PullmanME and Monroy GC. A naturally occurring inhibitor of mitochondrial adenosine triphosphatase. J Biol Chem 238: 3762–3769, 1963.[Free Full Text]
  35. ReimerKA. The slowing of ischemic energy demand in preconditioned myocardium. Ann NY Acad Sci 793: 13–26, 1996.[Medline]
  36. ReimerKA, Vander Heide RS, and Jennings RB. Ischemic preconditioning slows ischemic metabolism and limits myocardial infarct size. Ann NY Acad Sci 723: 99–115, 1994.[Web of Science][Medline]
  37. RosingJ, Harris DA, Kemp A Jr, and Slater EC. Nucleotide-binding properties of native and cold-treated mitochondrial ATPase. Biochim Biophys Acta 376: 13–26, 1975.[Medline]
  38. RouslinW. Regulation of the mitochondrial ATPase in situ in cardiac muscle: role of the inhibitor subunit. J Bioenerg Biomembr 23: 873–888, 1991.[CrossRef][Web of Science][Medline]
  39. RouslinW. Protonic inhibition of the mitochondrial oligomycin-sensitive adenosine 5'-triphosphatase in ischemic and autolyzing cardiac muscle. J Biol Chem 258: 9657–9661, 1983.[Abstract/Free Full Text]
  40. RouslinW and Broge CW. IF1 function in situ in uncoupler-challenged ischemic rabbit, rat, and pigeon hearts. J Biol Chem 271: 23638–23641, 1996.[Abstract/Free Full Text]
  41. RuiterJH, Spaan JA, and Laird JD. Transient oxygen uptake during myocardial reactive hyperemia in the dog. Am J Physiol Heart Circ Physiol 235: H87–H94, 1978.[Abstract/Free Full Text]
  42. ScholzTD and Balaban RS. Mitochondrial F1-ATPase activity of canine myocardium: effects of hypoxia and stimulation. Am J Physiol Heart Circ Physiol 266: H2396–H2403, 1994.[Abstract/Free Full Text]
  43. SchulzR, Cohen MV, Behrends M, Downey JM, and Heusch G. Signal transduction of ischemic preconditioning. Cardiovasc Res 52: 181–198, 2001.[Free Full Text]
  44. TakeoS and Nasa Y. Role of energy metabolism in the preconditioned heart—a possible contribution of mitochondria. Cardiovasc Res 43: 32–43, 1999.[Abstract/Free Full Text]
  45. TomasetigL, Di Pancrazio F, Harris DA, Mavelli I, and Lippe G. Dimerization of F0F1 ATP synthase from bovine heart is independent from the binding of the inhibitor protein IF1. Biochim Biophys Acta 1556: 133–141, 2002.[Medline]
  46. TuneJD, Richmond KN, Gorman MW, and Feigl EO. KATP channels, nitric oxide, and adenosine are not required for local metabolic coronary vasodilation. Am J Physiol Heart Circ Physiol 280: H868–H875, 2001.[Abstract/Free Full Text]
  47. Vander HeideRS, Hill ML, Reimer KA, and Jennings RB. Effect of reversible ischemia on the activity of the mitochondrial ATPase: relationship to ischemic preconditioning. J Mol Cell Cardiol 28: 103–112, 1996.[CrossRef][Web of Science][Medline]
  48. VuorinenK, Ylitalo K, Peuhkurinen K, Raatikainen P, Ala-Rämi A, and Hassinen IE. Mechanisms of ischemic preconditioning in rat myocardium. Roles of adenosine, cellular energy state, and mitochondrial F0F1-ATPase. Circulation 91: 2810–2818, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. Comelli, G. Metelli, and I. Mavelli
Downmodulation of mitochondrial F0F1 ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug
Am J Physiol Heart Circ Physiol, February 1, 2007; 292(2): H820 - H829.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/H2192    most recent
00327.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Penna, C.
Right arrow Articles by Mavelli, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Penna, C.
Right arrow Articles by Mavelli, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.