Phosphate-induced efflux of adenine nucleotides from rat-heart mitochondria: evaluation of the roles of the phosphate/hydroxyl exchanger and the dicarboxylate carrier.

Upon the addition of inorganic phosphate, isolated rat-heart mitochondria released endogenous adenine nucleotides. To elucidate the mechanism of this phosphate-induced efflux, we evaluated the relative roles of three inner mitochondrial membrane carriers: the adenine nucleotide translocase, the phosphate/hydroxyl exchanger, and the dicarboxylate carrier. Atractyloside (a specific inhibitor of the adenine nucleotide translocase) prevented this efflux, but did not inhibit mitochondrial swelling. Inhibitors of the phosphate/hydroxyl exchanger (200 microM n-ethylmaleimide and 10 microM mersalyl) did not inhibit phosphate-induced efflux. 200 microM mersalyl (which inhibited both the phosphate/hydroxyl exchanger and the dicarboxylate carrier) inhibited the rate of efflux approx. 65% Phenylsuccinate and 2-n-butylmalonate (inhibitors of the dicarboxylate carrier) partially inhibited phosphate-induced efflux and adenine nucleotide translocase activity. Mersalyl (200 microM) had no effect on adenine nucleotide translocase activity. Partial inhibition of the adenine nucleotide translocase by phenylsuccinate and butylmalonate could not explain the extent of inhibition of phosphate-efflux by these agents. Moreover, the rates of adenine nucleotide efflux in the presence of phenylsuccinate, butylmalonate, or mersalyl correlated well with the ability of these agents to inhibit succinate-supported respiration. We conclude that phosphate-induced efflux of adenine nucleotides from rat heart mitochondria occurs over the adenine nucleotide translocase, and that the site of action of the phosphate is not the phosphate/hydroxyl exchanger, but is likely the dicarboxylate carrier.

Attenuation of postischaemic dysfunction by ischaemic preconditioning is not mediated by adenosine in the isolated rat heart.

OBJECTIVE: The aim was to test the hypothesis that adenosine mediates the cardioprotective effects of ischaemic preconditioning in the isolated rat heart. METHODS: Transient exposure of the hearts to adenosine and the A1 selective agonist, PIA, were tested for the ability to mimic the cardioprotective effects of ischaemic preconditioning in hearts that underwent 40 min normothermic ischaemic followed by 30 min reperfusion. Treated hearts were perfused with 10 or 50 microM adenosine or 10(-7) M R-phenylisopropyladenosine (PIA) for 5 min followed by a 5 min washout period. Preconditioned hearts underwent 5 min of ischaemia and 5 min of reflow prior to the 40 min ischaemic period. The ability of the adenosine receptor antagonist, BW A1433U, to inhibit the cardioprotective effects of ischaemic preconditioning was also tested. The effects of these treatments on metabolite levels and postischaemic haemodynamic function were assessed. RESULTS: Adenosine (50 microM), but not PIA, resulted in enhanced accumulation of lactate after 40 min ischaemia: 122(SEM 8) v 96(5) nmol.mg-1 protein in control hearts (p < 0.002). Adenosine and PIA treatments did not significantly affect myocardial acidosis during ischaemia. Postischaemic contractile function (as assessed by percent recovery of the heart rate x developed pressure) was lower in 50 microM, but not 10 microM, adenosine treated hearts [8.8(2.2)] and PIA treated hearts [11.9(2.5)] than in control hearts [20.4(3.6)] (p < 0.01). Ischaemic preconditioning (1) lowered glycogen levels prior to the 40 min ischaemic period [57(6) v 110(18) nmol glucosyl units.mg-1 protein; p < 0.01]; (2) lowered lactate levels at the end of the 40 min ischaemic period [61(4) v 104(5) nmol.mg-1 protein]; (3) preserved myocardial pH during ischaemia [6.69(0.07) v 6.40(0.07); p < 0.01]; and (4) enhanced recovery of postischaemic contractile function [42.3(4.4)% v 19.7(6.0)%; p < 0.02]. BW A1433U did not prevent these effects of ischaemic preconditioning. CONCLUSIONS: The cardioprotective effects of ischaemic preconditioning are not mediated by adenosine released during the preconditioning period in the isolated rat heart. Also, transient treatment of the heart with A1 adenosine receptor agonists can exacerbate postischaemic contractile dysfunction.

Transient beta adrenergic stimulation can precondition the rat heart against postischaemic contractile dysfunction.

OBJECTIVE: The aim was to assess the abilities of exogenous noradrenaline, isoprenaline, and phenylephrine to precondition the isolated rat heart against ischaemic and reperfusion injury. METHODS: The isovolumetric Langendorff rat heart model was used to determine postischaemic recovery of left ventricular function. The hearts were subjected to 30 min of normothermic global ischaemia followed by 30 min reperfusion. Treated hearts were perfused with noradrenaline (10(-7) M), isoprenaline (10(-8) M), or phenylephrine (10(-6) M, 10(-5) M, and 10(-4) M) for 5 min followed by 5 min washout before the 30 min ischaemic period. RESULTS: Control hearts recovered 47.6(SEM 4.3)% of baseline heart rate x developed pressure after 30 min reperfusion, whereas noradrenaline and isoprenaline treated hearts recovered 75.1(4.6) and 76.4(4.6)%, respectively (p < 0.001 v control). Left ventricular end diastolic pressures at the end of reperfusion were 48.8(4.0), 20.0(2.4), and 21.6(2.7)mm Hg for control, noradrenaline treated (p < 0.001 v control), and isoprenaline treated (p < 0.001 v control) hearts respectively. beta Blockade with propranolol during noradrenaline treatment blocked the protective effects. No concentration of phenylephrine used was able to enhance postischaemic heart rate x developed pressure significantly, or result in improved (lower) postischaemic left ventricular end diastolic pressure. During treatment with noradrenaline and phenylephrine (10(-5) M), lactate release was 13.0(1.0) and 11.0(0.9) mumol.5 min-1, respectively (p = NS); these values were significantly (p < 0.001) greater than baseline value of 3.7(0.5) mumol.5 min-1. Immediately before the 30 min ischaemic period, control and phenylephrine treated groups had glycogen levels of 132(14) and 128(5) nmol.mg-1 protein, respectively (p = NS), whereas the glycogen content of the noradrenaline treated group was only 96(5) nmol.mg-1 protein (p < 0.05 v control and phenylephrine treated). CONCLUSIONS: Transient beta adrenergic but not alpha 1 adrenergic stimulation can precondition the isolated perfused rat heart. The mechanism of protection may, at least in part, be due to transient demand ischaemia. Partial depletion of glycogen following treatment may play a role in the observed protective effects.

Hypotension and cardiac stimulation due to the parathyroid hormone-related protein, humoral hypercalcemia of malignancy factor.

Patients with humoral hypercalcemia of malignancy display markedly increased serum calcium levels, reduced blood pressure, and tachycardia. The causative agent, humoral hypercalcemia of malignancy factor [also called PTH-related protein (PTHrp)] has been shown to interact with PTH receptors in bone and kidney. We compared human PTHrp-(1-34) with rat PTH-(1-34) for the effects of each peptide on cardiovascular function in unrestrained conscious rats. Both PTHrp and PTH decreased blood pressure in a dose-dependent manner over the concentration range of 0.3-30 micrograms/kg. PTHrp was approximately 3-fold more potent than PTH, producing up to a 50 mm Hg decrease in pressure within 2 min at 10 micrograms/kg. Both peptides increased heart rate more than 70 beats/min at this dose. However, PTH appeared to exert greater efficacy and potency than PTHrp in increasing heart rate in vivo. In the isolated and perfused rat heart, PTHrp and PTH produced positive chronotropic and positive inotropic effects as well as increased coronary flow. PTHrp was more potent and more effective than PTH. The time courses of these effects in the perfused heart preparations indicated that both peptides produced maximal effects within 1 min, with all responses returning to baseline within 10 min. In isolated helical strips of rat aorta, PTHrp and PTH relaxed norepinephrine-contracted tissues in a concentration-dependent fashion. A functional endothelium was not required for the relaxing effects of either peptide. These studies indicate that PTHrp and PTH decrease blood pressure by relaxing vascular tissue in an endothelium-independent manner. Also, these peptides directly increased heart rate, contractility, and coronary flow. Since PTHrp has recently been found in normal human cells, these studies suggest the possibility of PTHrp as a regulator or modulator of cardiovascular function.

Effect of increasing volume of cardioplegic solution on postischemic myocardial recovery.

Multidose cardioplegia has been reported to be superior to single-dose cardioplegia in protecting the heart during ischemia. However, large volumes of cardioplegic solution may be detrimental because of washout of adenine nucleotide degradation products that accumulate during ischemia, which limits recovery of adenosine triphosphate. We designed an experiment to test the effects of increasing the volume of cardioplegic solution on postischemic myocardial recovery. Four groups were studied: Group 1, initial 2 minute single dose of cardioplegic solution; Group 2, infusion of cardioplegic solution every 30 minutes for 1 minute; Group 3, infusion of cardioplegic solution every 20 minutes for 1 minute; and Group 4, infusion of cardioplegic solution every 20 minutes for 2 minutes. All groups were ischemic for 2 hours at 20 degrees C. Although washout of nucleotide degradation products during the ischemic interval increased with higher volumes of cardioplegic infusion, the total washout (infusion plus initial 5 minutes of reperfusion) was not different among all groups. The multidose groups recovered function better and had significantly higher levels of total tissue purines after 30 minutes of reperfusion. There was no difference in adenosine triphosphate levels among all groups after reperfusion. We conclude that increasing the volume of cardioplegic solution, within a clinically relevant range is not associated with increasing loss of adenine nucleotides from the cell or with impaired functional recovery of the heart.

Recovery of nonbeating donor hearts.

BACKGROUND: Use of hearts from non-heart-beating donors could greatly increase the pool of cardiac homografts. This hypothesis was investigated in a model of traumatic death with New Zealand rabbits which were exsanguinated after 10 minutes of hypotension. METHODS: The hearts were left in situ at normothermia until just before the onset of contracture, were surgically exposed, given either blood cardioplegic, crystalloid cardioplegic, or University of Wisconsin cardioplegic solution, and then stored for 1 hour at 4 degrees C in the respective solutions. The hearts were reperfused for 20 minutes with a Langendorff apparatus. A balloon was placed in the left ventricle, and peak developed pressure and diastolic pressure-volume relationship data were collected over a range of balloon volumes. Control data was obtained from nonischemic rabbit hearts. RESULTS: Blood cardioplegic solution preserved peak developed pressure and the slope of diastolic pressure-volume relationship at control levels, whereas crystalloid cardioplegic and University of Wisconsin solutions showed significant deterioration in both of these indexes. The total adenine nucleotide pool was significantly improved with the use of blood cardioplegic solution compared with crystalloid cardioplegic and University of Wisconsin solutions although all groups were significantly depressed compared with control values. CONCLUSIONS: This study indicates that hearts harvested from non-heart-beating donors can have function that is not statistically different from controls if preserved with blood cardioplegic solution and that University of Wisconsin and crystalloid cardioplegic solutions are inadequate for preserving hearts harvested under these unique conditions.

Heat shock improves recovery and provides protection against global ischemia after hypothermic storage.

BACKGROUND: Improved methods of donor heart preparation before preservation could allow for prolonged storage and permit remote procurement of these organs. Previous studies have shown that overexpression of heat-shock protein 72 provides protection against ischemic cardiac damage. We sought to determine whether rats subjected to heat stress with only 6-hour recovery could acquire protection to a subsequent heart storage for 12 hours at 4 degrees C. METHODS: Three groups of animals (n = 10 each) were studied: control, sham-treated, and heat-shocked rats (whole-body hyperthermia 42 degrees C for 15 minutes). After 12-hour cold ischemia hearts were reperfused on a Langendorff column. To confirm any differences in functional recovery, hearts were then subjected to an additional 15-minute period of warm global ischemia after which function and lactate dehydrogenase enzyme leakage were measured. RESULTS: Heat-shocked animals showed marked improvements compared with controls in left ventricular developed pressure (63+/-4 mm Hg versus 44+/-4 mm Hg, p<0.05) heart rate x developed pressure (13,883+/-1,174 beats per minute x mm Hg versus 8,492+/-1,564 beats per minute x mm Hg, p<0.05), rate of ventricular pressure increase (1,912+/-112 mm Hg/second versus 1,215+/-162 mm Hg/second, p<0.005), rate of ventricular pressure decrease (1,258+/-89 mm Hg/second versus 774+/-106 mm Hg/second, p<0.005). Diastolic compliance and lactate dehydrogenase release were improved in heatshocked animals compared with controls and sham-treated animals. Differences between heat-shocked animals and control or sham-treated animals were further increased after the additional 15-minute period of warm ischemia. Western blot experiments confirmed increased heat-shock protein 72 levels in heat-shocked animals (>threefold) compared with sham-treated animals and controls. CONCLUSIONS: Heat shock 6 hours before heart removal resulted in marked expression of heat-shock protein 72 and protected isolated rat hearts by increased functional recovery and decreased cellular necrosis after 12-hour cold ischemia in a protocol mimicking that of heart preservation for transplantation. Protection was further confirmed after an additional 15-minute period of warm ischemia.

Transient ischemia cannot precondition the rabbit heart against postischemic contractile dysfunction.

BACKGROUND: The rat heart is preconditioned against postischemic contractile dysfunction by a brief period of transient ischemia before a prolonged ischemic period. However, the rabbit heart does not receive such cardio-protection from pretreatment with a single transient ischemia periods. We hypothesized that in the rabbit heart, a multiple cycle of transient ischemia is required to reach a threshold necessary to precondition against postischemic contractile dysfunction. METHODS: To test this hypothesis, we subjected isolated, perfused rabbit hearts to either one 5-minute transient ischemic period or three 5-minute transient ischemic periods followed by a 40-minute period of warm ischemia and 30 minutes of reperfusion. Control hearts (no pretreatment with transient ischemia) were examined simultaneously. Left ventricular developed pressure was measured with an intraventricular balloon. RESULTS: Postischemic recoveries (expressed as percent of preischemic values) of left ventricular developed pressure for the group with one ischemic period and the group with three ischemic periods were 43% +/- 5% (n = 5) and 38% +/- 6% (n = 6), respectively. These values were not significantly different from control values. CONCLUSIONS: Neither one nor three periods of transient ischemia protect the isolated, perfused rabbit heart from postischemic contractile dysfunction. Therefore, the rabbit heart may not have the capacity to be ischemically preconditioned against postischemic contractile dysfunction.

Cardioprotection by local heating: improved myocardial salvage after ischemia and reperfusion.

BACKGROUND: Previous studies have shown that expression of the inducible 70-kD heat-shock protein (HSP72) by whole-body hyperthermia is associated with protection against ischemia-reperfusion injury. To develop techniques for regional elevation of heat-shock proteins that prevent extracardiac sequelae during whole-body hyperthermia, we sought to determine if local heating of the heart in vivo provides protection against ischemia-reperfusion injury in the rat. METHODS: A thermal probe was used to locally heat rat hearts at two adjacent sites on the epicardial surface of the left ventricle. Rats were subjected to either 30 minutes of sham surgery (control; n = 10) or two local applications of the probe at 42.5 degrees to 43.5 degrees C for 15 minutes each (n = 9). After 4 hours, rats were subjected to 30 minutes of regional ischemia followed by 120 minutes of reperfusion. Hearts were removed and area at risk and infarct area were determined. RESULTS: Localized heat stress resulted in a significant limitation of infarct size in heat-treated animals versus controls (mean +/- standard error of the mean infarct area/area at risk = 4.3% +/- 0.85 versus 19.2% +/- 3.4%; p < 0.005). Western blot experiments confirmed elevated HSP72 expression in left (heated) and right (nonheated) ventricular samples from treated animals (n = 6; left ventricular = 5.5-fold; right ventricular = 3.7-fold) compared with sham-operated controls. Controls treated with the probe at 37 degrees C (n = 4) showed no increases in HSP72. CONCLUSIONS: Local heating of the heart is associated with elevated levels of HSP72 and improved myocardial salvage. The increase in expression of HSP72 is not limited to the heated region, but extends into nonheated regions of the heart as well. This may lead to the development of new techniques that improve methods of myocardial revascularization and heart transplantation procedures.

Preconditioning with dobutamine in the isolated rat heart.

The ability of dobutamine to precondition the isolated rat heart against postischemic contractile dysfunction was assessed. Hearts were perfused with varying concentrations of dobutamine for 5 min followed by a 5 min "washout" period and 30 min of global ischemia. The hearts were reperfused for 30 min to assess postischemic function. Dobutamine improved postischemic developed pressure, +dp/dt, heart rate x developed pressure, end diastolic pressure, and coronary flow in a concentration-dependent manner. The concentration of dobutamine showing the maximum protective effect was 10(-6)M. Propranolol administered with dobutamine significantly attenuated the protective effect. The results indicate that transient treatment with dobutamine can precondition the rat heart against ischemia/reperfusion injury. The mechanism of protection appears to involve beta-adrenergic stimulation.

Release of AMP and adenosine from rat heart mitochondria.

Release of AMP and adenosine from rat heart mitochondria was studied. The rate of appearance of extramitochondrial adenosine was independent of the extramitochondrial phosphate concentration between 5 and 20 mM. In the absence of exogenous, respiratory substrates or in the presence of glutamate/malate plus rotenone, the rate of appearance of adenosine was relatively low when phosphate was not added. The appearance of extramitochondrial AMP + adenosine was found to be directly proportional to the extra-mitochondrial phosphate concentration. Zn2+ (10 mM) decreased the rate of adenosine appearance by 90% and increased the rate of AMP appearance 6-fold. The mitochondrial preparations dephosphorylated exogenous AMP; this activity was inhibited by 10 mM Zn2+. We conclude that the adenosine appearing in the extramitochondrial space was not due to a direct release from the matrix, but instead was due to adenine nucleotide release with subsequent conversion to adenosine in the extramitochondrial space.

Hemodynamic action of calcitonin gene-related peptide in the isolated rat heart.

The effects of calcitonin gene-related peptide (CGRP) on heart rate, coronary flow, pressure development, and time to ischemic contracture were studied in the isolated, perfused rat heart. A bolus of CGRP (2640 pmols) caused significant increases in heart rate and coronary flow; these effects were sustained for at least five minutes after injection. The increase in coronary flow was independent of heart rate, since CGRP caused an increase in coronary flow in non-beating (potassium-arrested) hearts. The dose-response of CGRP was studied using five doses (65, 218, 658, 1320 and 2640 pmols) given as bolus injections. Although the increase in heart rate was apparently dose-dependent, significant increases above baseline were observed only with the two highest doses. In contrast, coronary flow increased significantly above baseline with the injection of all but the lowest dose of CGRP. Ten minutes after injection of CGRP, all hearts were made ischemic. The time to onset of ischemic contracture was approximately 11 minutes for those hearts that received 65 pmols of CGRP; however, for those hearts receiving all other doses of CGRP, the time to onset of contracture was approximately 8 minutes. We conclude that CGRP significantly decreases the resistance of the coronary vascular bed, and that it may be an important regulator of regional blood flow in the heart.

Cocaine cardiotoxicity differs markedly in isolated hearts of two strains of rats exhibiting phenotypic differences in sensitivity to seizures.

Isolated hearts from two strains of rats bred for sensitivity or resistance to amygdala kindling that also exhibit, in vivo, differential sensitivity to the cardiotoxicity of cocaine were studied. The goal was to determine if the differential cardiotoxic sensitivity was due, at least in part, to intrinsic strain-dependent differences in the heart. The Langendorff preparation was used (n=8 per strain). Hearts were perfused with increasing concentrations of cocaine (5 x 10(-6), 1 x 10(-5), 5 x 10(-5), 1 x 10(-4), and 5 x 10(-4) M) for 5 min with a 5 min washout between exposure to successive concentrations. Consistent with in vivo observations, hearts from genetically slow amygdala kindling rats (Slow) required lower cocaine doses to develop cardiac arrhythmias and arrest as compared to the hearts from genetically fast amygdala kindling rats (Fast). At 5 x 10(-5) M cocaine arrhythmias occurred in 38% (3/8) Slow and 0% Fast hearts. Five of 8 Slow hearts and none of 8 Fast hearts were arrested by 10(-4) M cocaine. Arrest in Fast hearts occurred only with 5 x 10(-4) M cocaine. Cocaine constricted coronary arteries (no significant difference between strains). On the other hand, coronary arteries of Slow but not Fast hearts dilated during cocaine washout after perfusion with all but the highest concentration of cocaine. We conclude that factors intrinsic to the heart and coronary artery influence the sensitivity or response of these structures to cocaine.

Uncoupling of oxidative phosphorylation in rat liver mitochondria by chloroethanols.

Chloroethanols are toxic chemicals used in industry and also formed as a result of the metabolism of several widely used halogenated hydrocarbons. The effect of 2-chloroethanol (CE), 2,2-dichloroethanol (DCE) and 2,2,2-trichloroethanol (TCE) on rat liver mitochondrial respiration was studied. Rat liver mitochondria were isolated in a medium consisting of 250 mM sucrose, 10mM Tris-HCl and 1 mM EDTA (pH 7.4). Mitochondrial respiration was determined with an oxygen electrode at 30 degrees C and the polarographic buffer consisted of 250 mM mannitol, 10 mM KCl, 10 mM K2HPO4, 5 mM MgCl2, 0.2 mM EDTA and 10 mM Tris-HCl (pH 7.4). With succinate as the respiratory substrate and using chloroethanols (150 mM), CE stimulated respiration by 28.2 +/- 6.5% and DCE by 202.7 +/- 8.2% while TCE inhibited mitochondrial respiration (greater than 95%). The effect of change in the concentration of chloroethanols on mitochondrial respiration was also studied. CE showed maximum stimulation at 600 mM (97.6%), DCE at 150 mM (202.6%) and TCE at 30 mM (313.6%). Respiratory stimulation was independent of mitochondrial protein concentration. Chloroethanols (optimal concentrations for respiratory stimulation with succinate) inhibited mitochondrial respiration when glutamate-malate was used as the respiratory substrate. Estimation of adenosine triphosphate (ATP) showed that chloroethanols inhibited the synthesis of ATP. These results indicate that chloroethanols stimulate mitochondrial respiration by uncoupling oxidative phosphorylation and that the uncoupling potency is proportional to the extent of chlorination at the beta-position of haloethanol.

Myocardial glycogen depletion cannot explain the cardioprotective effects of ischemic preconditioning in the rat heart.

The mechanism of ischemic preconditioning remains unknown. The role of glycogen depletion prior to prolonged ischemia was examined as a potential mechanism of ischemic preconditioning. The glycogen content of the rat heart varies in a 24-h rhythm. In a retrospective study, the relationships between the time of day the animals were sacrificed, pre-ischemic myocardial glycogen content, and post-ischemic functional recovery were assessed in non-conditioned and ischemically preconditioned hearts. The analyses were performed on previously published data (Asimakis et al.. 1992, 1993). After an equilibration perfusion, isolated rat hearts were given 40 min of global ischemia followed by 30 min of reperfusion. Preconditioned hearts received 5 min of ischemia followed by a 5-min recovery period prior to the 40-min ischemic period. Some of the hearts were freeze-clamped immediately prior to the 40-min ischemic period to determine pre-ischemic glycogen content. Pre-ischemic glycogen was higher in the morning than afternoon. The time of day correlated significantly with the pre-ischemic glycogen content of non-conditioned (r = 0.67; P < 0.005) and preconditioned (r = 0.79; P < 0.001) hearts. However, time of day did not correlate significantly with post-ischemic recovery of heart rate x developed pressure (HR x DP) on end-diastolic pressure (EDP) in either the non-conditioned or preconditioned hearts. The relationships were also assessed by subdividing the groups into either morning (a.m.) or afternoon (p.m.) hearts. The pre-ischemic glycogen content was lower in the non-conditioned-p.m. (n = 5) hearts compared to the non-conditioned-a.m. (n = 10) hearts (67.6 +/- 9.0 nu 128.1 +/- 13.3 nmol glucose/mg protein P < 0.005). However, there were no significant differences between p.m. (n = 13) and a.m. (n = 9) non-conditioned hearts with respect to post-ischemic recovery of HR x DP (20.6 +/- 4 nu 12.0 +/- 4% of baseline, respectively, P = N.S.). In contrast, preconditioned-p.m. (n = 6) and -a.m. (n = 7) had pre-ischemic glycogen contents of 49.6 +/- 6 and 76.6 +/- 5.0 nmol glucose/mg protein, respectively. These glycogen values were not significantly different from the non-conditioned-p.m. hearts (67.6 nmol/mg protein). However, post-ischemic recovery of HR x DP in the preconditioned-p.m. (n = 5) and -a.m. (n = 6) hearts were 54.6 +/- 5 and 51.4 +/- 8% of baseline, respectively (these values were significantly higher (P < 0.05) than the recovery for the non-conditioned-p.m. and -a.m. hearts). The results imply that the cardioprotection of ischemic preconditioning cannot be explained solely by myocardial glycogen depletion.

Myocardial ischemia: correlation of mitochondrial adenine nucleotide and respiratory function.

State 3 respiration of rat heart mitochondria decreased approximately 60% after 20 min normothermic in vitro ischemia. After 20 min ischemia, the levels of intramitochondrial adenine nucleotides (ATP + ADP + AMP) decreased to approximately 20% of control values, with a rapid loss between 10 and 20 min. Also, the exchangeable adenine pool of the mitochondria decreased 60% after 20 min ischemia. State 4 respiration was not affected by the ischemic insult. The adenine nucleotide translocase activities of mitochondria from control and ischemic hearts were too high to measure accurately. Therefore, the effects of ischemia on adenine nucleotide translocase activity could not be established. However, 1 microM carboxyatractyloside did not impair state 3 respiration of control mitochondria, but did inhibit the adenine translocase activity by at least 80%. Moreover, titration of state 3 respiration with carboxyatractyloside produced sigmoidal curves for mitochondria from control and ischemic tissue. State 3 respiration correlated well with the total mitochondrial adenine nucleotides and the exchangeable adenine pool (r = 0.63 and 0.78, respectively). Data collected from isolated perfused rat hearts also showed a good correlation between state 3 respiration and the exchangeable adenine nucleotides (r = 0.92). In this study, mitochondria were isolated from hearts that were either perfused, made ischemic for 30 min by aortic cross-clamp, or reperfused for 10 min after the aortic cross-clamp. The slopes and y-intercepts of the regression lines were similar for the in vitro ischemic and the perfusion studies. There was no significant difference between the effects of ischemia on the state 3 and uncoupled respiratory rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Intramitochondrial adenine nucleotides and energy-linked functions of heart mitochondria.

Isolated rabbit heart mitochondria were incubated with varying amounts of inorganic pyrophosphate in 250 mM sucrose to specifically decrease the pool size of endogenous adenine nucleotides. The endogenous adenine nucleotide content decreased by as much as 80% as a result of this treatment. Phosphorylating respiration (state 3) declined from about 340 to 180 nAtoms O . min-1 . mg protein-1 over the full range of intramitochondrial adenine nucleotides measured (approx 7.5-1.5 nmol/mg protein). Uncoupled and nonphosphorylating (state 4) rates of respiration were not greatly affected by adenine nucleotide depletion. Respiratory activity of the adenine nucleotide-depleted mitochondria was enhanced by addition of exogenous adenosine 5'-triphosphate (ATP). Partial depletion (approx 40%) of the intramitochondrial adenine nucleotides resulted in an impaired ability of heart mitochondria to retain Ca2+. Premature Ca2+ efflux was associated with organelle swelling and altered energy coupling. Exogenous ATP or adenosine 5'-diphosphate (ADP) added prior to Ca2+ efflux restored Ca2+ retention in these mitochondria. Atractyloside inhibited the restoration of Ca2+ retention. This study indicates a significant role for endogenous adenine nucleotides in maintaining oxidative phosphorylation and Ca2+ transport in heart mitochondria. The results are discussed with regard to significance in ischemic heart damage.