Effect of prostaglandins I2 (prostacyclin) and F2 alpha on function, energy metabolism, and calcium uptake in ischaemic/reperfused hearts.

OBJECTIVE: The aim was to examine the effect on cardiac function, energy metabolism, and calcium uptake of either prostaglandin I2 (PGI2, prostacyclin) or prostaglandin F2 alpha (both 28.6 nM) on the response of isolated rat hearts to 25 min of total global ischaemia with or without 30 min reperfusion. METHODS: Rat hearts were perfused by the Langendorff method and function assessed by left ventricular pressure. Energy metabolites were measured using enzymatic techniques and 45Ca2+ uptake determined by radioisotopic analysis. RESULTS: Although there was no effect of either prostaglandin on contractile depression during ischaemia, both compounds accelerated the onset of and increased the magnitude of ischaemic contracture. High energy phosphate content at the end of the ischaemic period was not affected by prostaglandin treatment; however, tissue lactate levels were increased by PGI2 as was tissue calcium content. Under control conditions mean recovery of left ventricular developed pressure ranged from 66% to 83%. In the presence of PGI2 and PGF2 alpha, recovery of developed pressure was reduced to 20% and 38% of preischaemic values, respectively. The reduced recovery in developed pressure was accompanied by an approximately threefold increase in diastolic pressure (p < 0.05). The depression of functional recovery in reperfused hearts treated with prostaglandins was associated with various disturbances of cellular metabolism including depressed ATP and creatine phosphate content and increased tissue lactate and calcium following 30 min of reperfusion. A significant correlation was found between the changes in developed pressure and diastolic pressure during reperfusion and the reduction in ATP and creatine phosphate repletion. The deficit in recovery of ventricular function also correlated significantly with increased lactate and calcium accumulation in the reperfused heart. CONCLUSIONS: Low concentrations of PGI2 and PGF2 alpha can depress contractile recovery of the globally ischaemic heart through a mechanism associated with altered cellular energy metabolism and increased calcium accumulation.

Fatty acids suppress recovery of heart function after hypothermic perfusion.

Working rat hearts were perfused for 15 minutes at 37 degrees C before switching to a Langendorff perfusion (60 mm Hg aortic pressure) at 10 degrees C for 40 minutes of hypothermic arrest. Ventricular function was allowed to recover for 15 minutes at 37 degrees C by reestablishing the prehypothermic conditions. The perfusate was Krebs-Henseleit bicarbonate buffer containing 3% bovine serum albumin and either glucose (11 mmol/L) or glucose (11 mmol/L) plus palmitate (1.2 mmol/L) and gassed with 95% O2 and 5% CO2. In hearts receiving glucose alone as substrate, coronary flow was maintained constant during the 40 minutes of hypothermic arrest and returned to prehypothermic rates with rewarming. Ventricular function, as estimated by peak systolic pressure and heart rate, recovered to the prehypothermic level. When palmitate was added, coronary flow decreased continuously throughout the hypothermic perfusion (22% decrease by 40 minutes), and ventricular pressure development was lower throughout the rewarming perfusion. Tissue levels of adenosine triphosphate and creatine phosphate were well maintained and long-chain acyl coenzyme A and acyl carnitine decreased during hypothermia regardless of the substrate provided. With rewarming, tissue levels of adenosine triphosphate and creatine phosphate decreased in those hearts receiving palmitate. Omission of fatty acid either during hypothermia or during the first 5 minutes of rewarming improved recovery of function. Addition of oxfenicine to inhibit fatty acid oxidation, or inhibition of Ca2+ overload by verapamil and low perfusate Ca2+, prevented the effects of palmitate on ventricular function.(ABSTRACT TRUNCATED AT 250 WORDS)

Deleterious effects of digitalis on reperfusion-induced arrhythmias and myocardial injury in ischemic rat hearts: possible involvements of myocardial Na+ and Ca2+ imbalance.

Isolated rat hearts were made ischemic for 25 min after an initial recirculating perfusion, followed by 30 min of reperfusion. In some hearts, interventions including administration of ouabain and/or high [K+] in the buffer were performed during the first 10 min of reperfusion. During ischemia, intracellular Na+ (Nai) increased from 15 to 64 mumol/g dry weight (dwt). During reperfusion, Nai declined rapidly (at 10 min of reperfusion: 48 mumol/g dwt, at 30 min: 25 mumol/g dwt) and regular rhythm was recovered within 10 min in hearts without any intervention during reperfusion. 45Ca2+ uptake increased from 0.8 to 7.5 mumol/g dwt after 30 min of reperfusion. Ventricular function recovered by 45%. A 10-min perfusion with 10 or 50 microM of ouabain increased Nai (17 to 21 or 27 mumol/g dwt) with increased left-ventricular (LV) contractile function, but these effects were reversed by combination of high perfusate [K+] (20 mM) in non-ischemic hearts. A 10-min reperfusion with ouabain retarded or stopped the decline in Nai (at 10 min of reperfusion: 54 or 63 mumol/g dwt, at 30 min: 32 or 40 mumol/g dwt). These amounts of ouabain also increased the incidence of ventricular tachyarrhythmias during reperfusion to 30% or 50%, and increased the duration of ventricular fibrillation from 6.5 to 11.5 or 18.0 min. 45Ca2+ uptake reached to 8.8 or 10.0 mumol/g dwt, and function recovered only 35% or 28%. When high perfusate [K+] was combined with ouabain during reperfusion, the retarded decline in Nai, augmented 45Ca2+ uptake, and reduced recovery of function caused by ouabain alone were attenuated. These results suggest that digitalis has toxic effects on reperfused ischemic hearts by inhibition of rapid active outward transport of previously elevated Nai and potentiation of Ca2+ overload.

[Enhancement of susceptibility to ouabain in ischemic rat heart].

During 10 mins of reperfusion after 25 mins global ischemia, subtoxic doses of ouabain (50, 100 microM) were used and followed by 20 mins reperfusion with standard buffer. At these doses ouabain had no harmful effects with 29% and 45% increase in developed pressure in aerobic hearts. Intracellular Na+ (Nai), 45Ca2+ uptake and recovery of ventricular function were measured. Nai increased from 15 to 64 mumol/g dw with no increase in 45Ca2+ uptake during ischemia. Upon reperfusion with standard buffer, additional gain in Nai at 2 mins (73 mumol/g dw) was followed by a rapid decline (at 10 mins: 48 mumol/g dw). 45Ca2+ uptake increased from 0.8 to 7.5 mumol/g dw after 30 mins reperfusion with decreased recovery of function (45%) and increased LVEDP (29 mmHg). Reperfusion with ouabain accelerated initial rise in Nai (2 mins: 79 and 83 mumol/g dw) and decline of Nai was retarded (10 mins: 65 and 83 mumol/g dw). Consequently, 45Ca2+ uptake and depression of function were augmented (Ca: 10.0, 11.5 mumol/g dw; function: 27%, 18%; LVEDP: 47, 48 mmHg) even when hearts were switched back to standard buffer. Combination of high K+ (20mM) reversed the effect of ouabain. The results suggested increased susceptibility to ouabain was caused by inhibited outward Na+ transport resulting in enhanced Ca2+ influx through Na+/Ca2+ exchange.

Lipid accumulation in the perfused rat heart after isoproterenol administration.

Isoproterenol is a potent lipolytic agent. It has also been shown to induce accumulation of lipid droplets in the myocardium in vivo. The isolated working rat heart model was applied to evaluate the effect in vitro. Twenty-four rats were randomly separated into four experimental groups of six rats, all receiving Krebs-Hensleit buffer with 11mM glucose. The control group had no supplement, the other groups received either 10(-5) M isoproterenol, 1.2 mM palmitate, or a combination of the two. Tissue levels of triglycerides and phospholipids, and the fractional volume of lipid droplets were determined at the end of the 30 min perfusion in the working heart mode. Ventricular function was monitored continuously and showed a marked increase in the isoproterenol treated groups. The level of tissue phospholipids remained unchanged in all groups. Whereas tissue triglycerides were significantly increased in the group receiving only palmitate as compared to the other groups. A corresponding increase was, however, not found to be significant for lipid droplet quantitation. The results are discussed in terms of possible mechanisms for isoproterenol-induced accumulation of neutral lipids in the myocardium.

Intermittent perfusion of ischemic myocardium. Possible mechanisms of protective effects on mechanical function in isolated rat heart.

Intermittent restoration of coronary flow during ischemia reduced myocardial damage and improved recovery of function. The mechanisms of the protective effects of intermittent perfusion were investigated in isolated rat hearts. Ventricular function was assessed as the product of developed pressure (left ventricular systolic pressure minus end-diastolic pressure) and heart rate. Recovery of function was calculated by division of the product at the end of reperfusion by that before ischemia. After 40 minutes of sustained global ischemia, intracellular Na+ (Nai) increased from 11 to 74 mumol/g dry wt. During 30 minutes of reperfusion, these hearts took up a large amount of 45Ca2+ (10 mumol/g dry wt), recovered only 24% of preischemic function, and had an increased left ventricular end-diastolic pressure (48 mm Hg). When the 40-minute period of ischemia was interrupted at 10-minute intervals by intermittent perfusion (three periods of 3 minutes) with either oxygenated or hypoxemic buffer, Nai increased to only 12 or 17 mumol/g dry wt, and reperfusion resulted in much lower 45Ca2+ uptake (0.5 and 0.5 mumol/g dry wt, respectively). Recovery of function was 100% of the preischemic value. When hypoxemic buffer without glucose was used for intermittent perfusion, Nai increased to 50 mumol/g dry wt, ATP was depleted, and reperfusion resulted in reduced recovery of function (76%) and moderately increased 45Ca2+ uptake (2.1 mumol/g dry wt). The role of Na(+)-K+ pump activity in maintaining low Nai was assessed by removing K+ from oxygenated or hypoxemic buffers used during intermittent perfusion. Under these conditions, Nai rose to 64 or 102 mumol/g dry wt, 45Ca2+ uptake increased to 4.4 or 9.4 mumol/g dry wt, and recovery of function was poor. There was a highly significant correlation between Nai during ischemia and reperfusion Ca2+ overload (r = 0.87) or impaired recovery of function (r = 0.96). These results indicate that prevention of an increase in Nai by maintenance of Na(+)-K+ pump activity is associated with a reduction of Ca2+ overload through Na+/Ca2+ exchange.

Mechanisms of reduced reperfusion injury by low Ca2+ and/or high K+.

Mechanisms of the protective effects of low Ca2+ (0.15 mM) and/or high K+ (20 mM) concentrations in the buffer on reperfusion injury were investigated. Intracellular Na+ (Nai+) increased fourfold during 25 min of ischemia. When hearts were reperfused with the standard buffer (1.25 mM Ca2+, 5.9 mM K+), Nai+ increased further during the 1st 2 min (5-fold) and then declined by 30% at 10 min of reperfusion. Ca2+ uptake increased 6- and 12-fold at 10 and 30 min of reperfusion, respectively. Function, which was assessed as the product of developed pressure and heart rate, recovered to 45% of the preischemic value and end-diastolic pressure was elevated (EDP: 31 mmHg). Reperfusion for 10 min with low Ca2+ buffer abolished the increase in Ca2+ uptake during this period, but it increased 10-fold when the perfusate was switched back to the standard buffer. Accelerated Ca2+ influx at this time was probably through Na(+)-Ca2+ exchange because Nai+ did not decline during low Ca2+ reperfusion. Elevation of EDP was suppressed (12 mmHg), but development of pressure did not increase. Reperfusion for 10 min with high K+ buffer accelerated the decline in Nai+ by 70% and reduced the increase in Ca2+ uptake (8-fold). Recovery of function improved (67%, EDP: 18 mmHg). Further improvement in function (78%, EDP: 10 mmHg) was obtained along with less Ca2+ uptake (7-fold) when low Ca2+ and high K+ were combined. Recovery of energy metabolites at 10 and 30 min of reperfusion was not different among the groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial metabolism of pantothenic acid in chronically diabetic rats.

Transport and metabolism of [3H]pantothenic acid ([3H]Pa) was investigated in hearts from control and streptozotocin-induced diabetic rats. In isolated perfused hearts from control animals, the transport of [3H]Pa was linear over 3 h of perfusion when 11 mM glucose was the only exogenous substrate. The in vitro transport of [3H]Pa by hearts from 48-h diabetic rats was reduced by 65% compared to controls and was linear over 2 h of perfusion with no further accumulation of Pa during the third hour. The defect in transport observed in vitro could be corrected by in vivo treatment with 4 U Lente insulin/day for 2 days. In vitro addition of insulin in the presence of 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate had no effect on [3H]Pa transport in hearts from 48-h diabetic rats during 3 h of perfusion. Accumulation of [3H]Pa was not inhibited by inclusion of 0.7 mM amino acids, 1 mM carnitine, 50 microM mersalic acid or 1 mM panthenol, pantoyllactone or pantoyltaurine. Uptake was inhibited by 1 mM nonanoic, octanoic or heptanoic acid, 0.1 mM biotin or 0.25 mM probenecid, suggesting a requirement for the terminal carboxyl group for transport. Transport of pantothenic acid was reduced in hearts from diabetic rats within 24 h of injection of streptozotocin. In vitro accumulation of [3H]Pa decreased to 10% of control 1 week after streptozotocin injection and then remained at 30% of the control value over 10 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular washout reduces Ca2+ overload and improves function of reperfused ischemic hearts.

Relationships between myocardial Ca2+ uptake, recovery of ventricular function, and restoration of tissue metabolites were determined during 30 min of reperfusion following ischemic and anoxic perfusion with either zero or low coronary flow, zero flow with intermittent perfusion, and low-flow perfusion without substrates. When zero-flow ischemia was maintained for 30 or 40 min, tissue lactate levels increased approximately 100-fold; with reperfusion of these hearts, developed pressure recovered to only 70 and 40% of preischemic function, respectively, and Ca2+ uptake increased by 7- and 15-fold. In contrast, 30 min of low-flow (1 ml/min) anoxic perfusion resulted in accumulation of less lactate (15-fold increase), less reperfusion Ca2+ uptake, and recovery of developed pressure to the preanoxic level. Omission of energy substrates during the low-flow anoxic perfusion caused a reduced recovery of heart rate with lower high-energy phosphate levels and increased Ca2+ uptake, but contractile function recovered to the same extent as in low-flow perfusion with substrate. Even very low flow rates (0.06-0.16 ml/min) of oxygen-deficient perfusate increased high-energy phosphate content and contractile function and decreased Ca2+ uptake. Intermittent perfusion with either oxygenated or anoxic buffer between four 10-min episodes of ischemia reduced lactate accumulation, maintained function, and left Ca2+ uptake essentially unchanged. Recovery of developed pressure during reperfusion was negatively correlated with the amount of lactate that accumulated during ischemia or anoxia and with reperfusion Ca2+ uptake, regardless of the duration or type of ischemia or anoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Na+ accumulation increases Ca2+ overload and impairs function in anoxic rat heart.

Maintenance of low coronary flow (1 ml/min) during 40 or 70 min of anoxia maintained function and prevented Ca2+ overload during reoxygenation in isolated rat hearts. In comparison, recovery from 40 min of global ischemia resulted in only 20% of preischemic function and an increase in end-diastolic pressure (LVEDP) to 39 mmHg. Reperfusion Ca2+ uptake rose from 0.6 to 10.2 mumol/g dry tissue. Intracellular Na+ (Nai+) increased from 13 to 61 mumol/g dry tissue after 40 min of global ischemia, but was unchanged in hearts with low flow anoxia. When glucose and pyruvate were omitted from buffer used for anoxic perfusion, recovery was only 15% of preanoxic values, LVEDP rose to 32 mmHg, and reperfusion Ca2+ uptake was 7.2 mumol/g dry. In addition, Nai+ increased (47.4 mumol/g dry tissue) and ATP was depleted (1.0 mumol/g dry tissue) in the absence of substrate. In anoxic hearts supplied substrate, Nai+ stayed low (12 mumol/g dry tissue) and ATP was preserved (11.6 mumol/g dry tissue). Addition of ouabain (100 or 200 microM) and provision of zero-K+ buffer increased Nai+ and resulted in impaired functional recovery, increased LVEDP, and greater reperfusion Ca2+ uptake. These interventions also decreased energy availability in anoxic hearts. To distinguish between effects of Na+ accumulation and ATP depletion, monensin, a Na+ ionophore, was added during low flow anoxia. Monensin increased Nai+, decreased functional recovery and increased reperfusion Ca2+ uptake in a dose-dependent manner (1-10 microM) without changing ATP content. These results suggested that reduction of Nai+ accumulation by maintenance of Na+, K+ pump activity was the major mechanism of the beneficial effects of low coronary flow on reperfusion injury.

Role of intracellular Na+ in Ca2+ overload and depressed recovery of ventricular function of reperfused ischemic rat hearts. Possible involvement of H+-Na+ and Na+-Ca2+ exchange.

The roles of H+-Na+ and Na+-Ca2+ exchange in the depression of ventricular function were studied in the reperfused isolated ischemic rat heart. Zero-flow global ischemia was induced for either 15 or 30 minutes and was followed by 30 minutes of aerobic reperfusion. Intracellular Na+ (Na+i) and 45Ca2+ uptake were measured during ischemia and reperfusion. Accumulation of Na+i was modified by prior glycogen depletion and by treatment with amiloride, a H+-Na+ exchange inhibitor, or monensin, a Na+ ionophore. Na+i rose continuously during ischemia and rapidly during the first two minutes of reperfusion. The larger inhibitory effect of amiloride and preischemic glycogen depletion was on Na+i accumulation during reperfusion; this finding suggests that the uptake occurs by H+-Na+ exchange. Reduction of Na+i accumulation by glycogen depletion was associated with less lactate and, presumably, H+ production and accumulation during ischemia. The rapid increase in Na+i during early reperfusion may reflect the readjustment of the low intracellular pH resulting from ischemia. The level of Na+i at the end of ischemia and especially after two minutes of reperfusion were linearly correlated with 45Ca2+ uptake and depression of ventricular function during subsequent reperfusion. This highly significant correlation between Na+i and 45Ca2+ uptake when Na+i was varied by several independent procedures, including monensin, strongly suggests that reperfusion 45Ca2+ uptake occurs at least in part by Na+-Ca2+ exchange. The rate of 45Ca2+ uptake during reperfusion was linearly and highly significantly correlated with elevation of diastolic pressure, reduced developed pressure, and decreased recovery of ventricular function. The data strongly support a mechanism of ischemic cell damage that involves excessive production and accumulation of H+ during ischemia that exchanges for extracellular Na+ during ischemia and rapidly during the first few minutes of reperfusion. Increased Na+i then causes excessive 45Ca2+ uptake and depressed recovery of cellular functions with continued reperfusion. Increased levels of Na+i may be a major event that couples a decreased intracellular pH during ischemia to excessive 45Ca2+ uptake and depressed recovery of cellular function with reperfusion.

Metabolism of pantothenic acid in hearts of diabetic rats.

The metabolism of pantothenic acid (Pa) by cardiac muscle was studied in normal and diabetic rats. Tissue levels of Coenzyme A (CoA) are elevated in the heart during early (6 to 12 h) diabetes, remains at a high level for several days, and then returns to normal or below normal levels. The increase in total tissue CoA mainly occurs in myocytes as indicated by isolation of cardiac myocytes from control and diabetic animals and measuring their content of CoA. The CoA concentration increased from 37 to 93 microM in the cytosolic compartment and from 2.0 to 2.6 mM in the mitochondrial matrix. These effects of diabetes were reversed by insulin treatment. CoA synthesis in hearts removed from control rats and perfused in vitro was stimulated by including in the perfusate Pa, cysteine and dithiothreitol, but no exogenous energy substrate. This stimulated in vitro rate of CoA synthesis was reduced in hearts removed from diabetic animals, and the reduction increased with duration of diabetes. The reduced rate in diabetic hearts resulted from both a decreased rate of Pa phosphorylation and decreased Pa transport. Transport of Pa into myocytes was decreased by as much as 80% in hearts from diabetic animals. The low transport rate was due to a decrease in Vmax with no apparent change in Km. Treatment of the isolated heart with insulin did not correct the diabetic-induced reduction in Pa transport. The transport rate in normal and diabetic hearts was not influenced by the type of energy substrate provided to the heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic disturbances after coronary occlusion.

In addition to the loss of oxygen supply and oxidative metabolism, a lack of coronary flow causes abnormalities of glycolytic anaerobic metabolism and associated ion fluxes. During reperfusion, the accumulation of massive amounts of calcium appears to contribute significantly to irreversible myocardial damage. The implications of these observations are discussed.

Inhibition of post-ischemic ventricular recovery by low concentrations of prostacyclin in isolated working rat hearts: dependency on concentration, ischemia duration, calcium and relationship to myocardial energy metabolism.

The objective of this study was to characterize the effect of prostacyclin (PGI2) on ventricular function following total global ischemia in isolated working rat hearts and to investigate the mechanism of its action. Ischemia was initiated for 10, 15, 20 or 25 min with or without treatment with PGI2. Increasing durations of ischemia resulted in a progressive decline in high energy phosphate (HEP) stores, an elevation in tissue lactate, and incomplete recovery of function with reperfusion. Prostacyclin at either 1 or 10 ng/ml had no effect on HEP levels or total adenine nucleotides, and tissue lactate was not significantly affected by PGI2 in hearts made ischemic for 10 to 20 min, but both PGI2 concentrations significantly elevated lactate levels after 25 min ischemia. Reperfusion recovery of left ventricular function was complete following 10 and 15 min ischemia, but incomplete recovery was evident following 20 min ischemia (77% of pre-ischemic function); and although PGI2 had no direct effect on the function of aerobically perfused hearts, recovery of aortic flow with 1 ng/ml PGI2 after 20 min of ischemia was reduced to approximately 20% (P less than 0.01). This depression in recovery was associated with significantly increased lactate levels during reperfusion. At a concentration of 10 ng/ml PGI2 did not depress ventricular recovery or elevate lactate content after 20 min ischemia. When hearts made ischemic for 20 min were analyzed, a significant negative correlation was found between ventricular recovery (aortic flow rate) and lactate concentration; however, no correlation existed between recovery and ATP levels. After 25 min of ischemia, five of eight (62.5%) untreated hearts demonstrated some degree of ventricular recovery, however, only two of ten hearts studied demonstrated any measurable functional recovery with either PGI2 concentration. This effect of PGI2 to reduce or prevent recovery of ventricular function following either 20 or 25 min of ischemia as well as the corresponding elevation in lactate levels was prevented by treatment with the calcium channel blocker verapamil. This study therefore shows that PGI2 at critical low concentrations can depress left ventricular recovery following total ischemia. This effect of PGI2 becomes more pronounced as ischemia duration is prolonged and is associated with elevated tissue lactate levels. The studies with verapamil suggest that PGI2 may be acting via the slow calcium channel to increase lactate levels and depress ventricular recovery following prolonged periods of ischemia.

Hearts from diabetic rats are more resistant to in vitro ischemia: possible role of altered Ca2+ metabolism.

The effects of whole heart ischemia were studied in isolated perfused rat hearts from control and diabetic animals. When whole heart ischemia was maintained for 30 minutes at 37 degrees C, diabetic hearts recovered 100% whereas hearts from normal animals recovered 30% of their preischemic function. Reperfusion Ca2+ uptake was about 2.5 microM/g dry wt in diabetic hearts compared with 10 microM/g dry wt in control hearts. When the ischemic period was extended to 40, 50, and 60 minutes, diabetic hearts had depressed recovery of ventricular function, and greater Ca2+ overload but reperfusion function was still significantly higher and Ca2+ overload significantly less than in control hearts. Depressed function and increased Ca2+ uptake were both linearly related to low tissue levels of residual high energy phosphates and inversely related to the amount of lactate that accumulated in the tissue during ischemia. However, regression lines relating these metabolic changes to depressed function and increased Ca2+ uptake showed that for any level of residual high energy phosphate or ischemic lactate, diabetic hearts performed much better and had less Ca2+ uptake than control hearts. These effects of diabetes were due to the diabetogenic action of the drugs used since both streptozotocin and alloxan had the same effect and in vivo insulin treatment reversed the effect. Diabetic hearts had a reduced maximum inotropic effect to increased extracellular Ca2+ under control aerobic perfusion conditions. The improved recovery of ventricular function during reperfusion of ischemic hearts from diabetic animals was highly correlated with reduced Ca2+ uptake, and regression lines relating depressed ventricular function to Ca2+ overload showed that data from control and diabetic hearts fell on the same line; that is, when depressed function occurred it was related to increased Ca2+ uptake to the same extent in both control and diabetic hearts. The resistance to ischemia in diabetic hearts was not related to higher tissue levels of high energy phosphates during reperfusion nor to lactate accumulation during ischemia. The observations suggest a role of increased reperfusion Ca2+ influx in ischemic damage and that alterations of sarcolemmal Ca2+ transport systems in diabetic myocardium may account for the greater resistance of these hearts to ischemia.

Inhibition of myocardial lipase by palmityl CoA.

The lipase activity of the adult rat heart consists of at least two components; a lipoprotein lipase and a "hormone-sensitive" or triglyceride lipase. The control of the triglyceride lipase by intermediates of lipid metabolism was studied in rat heart homogenates. Perfusion of hearts with fatty acids, glucose or no exogenous substrate did not alter lipase activity. Bovine serum albumin (BSA) stimulated the in vitro lipase activity whereas palmityl-coenzyme A (CoA) was a potent inhibitor. Other fatty acid intermediates such as acetyl-CoA, acetyl-carnitine, palmityl-carnitine and palmitate had little or no effect. Long-chain acyl CoA may be an important intermediate for matching triglyceride hydrolysis with the supply of extracellular fatty acids and the rates of fatty acid oxidation.

Mitochondrial synthesis of coenzyme A is on the external surface.

Synthesis of coenzyme A (CoA) in isolated rat heart mitochondria was studied. Mitochondria synthesized CoA from 4'phosphopantetheine (4'PP), a precursor of CoA which is synthesized from pantothenic acid in the cytosol. The synthesis was time dependent and was absolutely dependent on the presence of external ATP under the conditions used. The rate of synthesis was increased either by increasing the pH from 7.4 to 8.5 or by adding 0.01% deoxycholate to the incubation medium. To determine whether the synthesis was intra- or extramitochondrial, mitochondria were separated from the incubation mixture by centrifugation and assayed for CoA. The amount of CoA appearing in the supernatant after 30 mins of incubation increased with increasing concentrations of 4'PP while that in the mitochondrial pellet remained constant over the concentration range studied. Synthesis of CoA from 4'PP was not affected by the uncoupler 2,4-dinitrophenol, or by carboxyatractyloside, or by a combination of the two drugs. The combination was used in an effort to deplete intramitochondrial ATP and to prevent external ATP from entering the mitochondria, thus resulting in mitochondria devoid of matrix ATP. The absolute dependence of synthesis on external ATP, the appearance of newly synthesized CoA in the incubation buffer and the ability of mitochondria lacking matrix-ATP to synthesize CoA suggest that the mitochondrial enzymes responsible for synthesis of CoA from 4'PP are on the outer membrane or on the outer side of the inner membrane.

A transport system for coenzyme A in isolated rat heart mitochondria.

The ability of isolated rat heart mitochondria to take up coenzyme A (CoA) from the incubation medium was studied. Mitochondria accumulated CoA in a time- and concentration-dependent manner. The accumulation process occurred in two phases. Within the first 30 s of incubation, mitochondrial content of CoA increased, and this phase did not plateau in the concentration range studied. Following this initial increase, a second slower phase of CoA accumulation occurred which plateaued around 50 microM CoA. The initial phase was decreased significantly by ATP or by carboxyatractyloside. In contrast, the presence of ATP or carboxyatractyloside did not affect the second phase. Decreasing the temperature from 30 to 0 degrees C did not affect the initial phase, but the second phase was almost abolished. In the presence of metabolic inhibitors (either 2,4-dinitrophenol or a combination of rotenone and antimycin), the initial "binding" phase was not affected; but the second "uptake" phase was abolished. These results suggest that the first phase of mitochondrial CoA accumulation is probably CoA binding to adenine recognizing sites on the mitochondria while the second phase may represent a specific uptake process for CoA which, although not directly ATP-dependent, is energy-dependent.

Stimulation of myocardial coenzyme A degradation by fatty acids.

Coenzyme A (CoA) levels were increased in isolated hearts from 537 +/- 14 to 818 +/- 44 nmol/g dry wt by perfusion for 45 min under conditions known to stimulate CoA synthesis (5). Subsequently, perfusion of these hearts with buffer containing glucose (11 mM) and pyruvate (5 mM) for 3 min had no effect on CoA levels (789 +/- 42 nmol/g dry wt). However, perfusion with a buffer containing glucose (11 mM) and palmitate (1.2 mM) decreased CoA levels to 683 +/- 34 nmol/g dry wt within 3 min. This decrease in CoA appeared to occur in the cytosolic compartment with no change in mitochondrial CoA content and was associated with a rise in tissue content of long-chain acyl-CoA. An increased incorporation of fatty acids into triglycerides was associated with the rise in total acyl-CoA suggesting that long-chain acyl-CoA levels were elevated in the cytosolic compartment. Perfusion conditions which maximally increased acyl-CoA levels also maximally stimulated CoA degradation. These observations suggest that the cytosolic degradation of CoA is related to high levels of long-chain acyl-CoA in this compartment. Use of these perfusion conditions in future studies should help define the pathway of CoA degradation and determine the mechanisms which control cellular levels of CoA.

Coenzyme A degradation in the heart: effects of diabetes and insulin.

Coenzyme A (CoA) degradation was studied in isolated working hearts from acutely diabetic rats (48 h). Hearts from diabetic rats had elevated levels of total CoA (752 +/- 15 nmol/g dry) compared to control (537 +/- 14 nmol/g dry). When hearts from diabetic animals were perfused for 5 mins with perfusate containing pyruvate, (5 mM) and glucose (11 mM) CoA levels remained unchanged. Addition of palmitate, (1.2 mM) and glucose (11 mM) to the perfusate, however, resulted in a rapid drop in CoA levels to 672 +/- 19 nmol/g dry. Palmitate had no effect on CoA levels in control hearts which did not have elevated levels of CoA. Addition of insulin to the buffer containing glucose and palmitate prevented the decrease in CoA levels in diabetic hearts. The level of long chain acyl CoA in diabetic hearts perfused with pyruvate was 105 +/- 11 nmol/g dry, and did not change when insulin was present in the perfusate. In the presence of palmitate, levels of long chain acyl CoA increased from 76 +/- 16 to 149 +/- 13 nmol/g dry, and, in this case, addition of insulin caused a further increase to 192 +/- 18 nmol/g dry. Thus, the lower rate of CoA degradation in the presence of insulin was associated with a rise in long chain acyl CoA levels. In a separate series of experiments, CoA levels were increased in control hearts in vitro (from 537 +/- 14 to 842 +/- 19 nmol/g dry). Subsequent perfusion of these hearts that contained elevated CoA with palmitate also resulted in a rapid drop of CoA to 655 +/- 17 nmol/g dry.(ABSTRACT TRUNCATED AT 250 WORDS)