Subcellular calcium transport during contractile failure and recovery in heart perfused with Na-free medium

Perfusion of isolated rat hearts with Na-free medium resulted in an immediate increase in contractile force followed by a decline and complete loss of contractile force within 25 s. The recovery of the contractile force upon reperfusion was only partial if the duration of Na-free perfusions was 10 min or longer. Ca binding and uptake activities of mitochondria obtained from hearts perfused with Na-free medium did not change significantly. However, Ca binding and uptake activities of microsomes were depressed after 5 min of perfusion. The critical concentration of Na in the perfusion medium for inducing these changes was found to be less than 35 mM. The microsomal Ca-ATPase activity was decreased after 10 min of Na-free perfusion. Only partial recovery of microsomal Ca uptake was observed upon reperfusion of hearts preperfused with Na-free medium for 20 min or longer whereas Ca-ATPase activity in these hearts did not recover at all. These results suggest that the defect in the microsomal Ca transport may be secondary to the development of contractile failure and may partially be associated with the inability of Na-depleted hearts to recover fully their contractile force.     

Reversibility of ultrastructural, contractile function and Ca2+ transport changes in guinea pig hearts after global ischemia

To understand the subcellular basis of contractile failure due to ischemia-reperfusion injury, effects of 20, 60, and 90 min of global ischemia followed by 30 min of reperfusion were examined in isolated guinea pig hearts. Cardiac ultrastructure and function as well as Ca2+ transport abilities of both mitochondrial and microsomal fractions were determined in control, ischemic, and reperfused hearts. Hearts were unable to generate any contractile force after 20 min of ischemia and showed a 75% recovery upon reperfusion. However, there were no significant changes in the subcellular Ca2+ transport in the 20-min ischemic or reperfused hearts. When hearts were made ischemic for 60 and 90 min, the recovery of contractile force on reperfusion was 50 and 7%, respectively. There was a progressive decrease in mitochondrial and microsomal Ca2+ binding and uptake activities after 60 and 90 min of ischemia; these changes were evident at various times of incubation period and at different concentrations of Ca2+. Mitochondrial Ca2+ transport changes were only partially reversible upon reperfusion after 60 and 90 min of ischemia, whereas the microsomal Ca2+ binding, uptake and Ca2+ ATPase activities deteriorated further upon reperfusion of the 90-min ischemic hearts. Ultrastructural changes increased with the duration of the ischemic insult and reperfusion injury was extensive in the 90-min ischemic hearts. These data show that the lack of recovery of contractile function upon reperfusion after a prolonged ischemic insult was accompanied by defects in sarcoplasmic reticulum Ca2+ transporting properties and structural damage.     

Modification of intramuscular pH oscillations in the isolated perfused rat heart by different interventions.

Changes in the intramuscular pH oscillations were examined by the use of an antimony electrode upon perfusing the isolated rat heart under different experimental conditions. The pH oscillations were decreased upon perfusing the hearts with Na+- or Ca2+-free medium and increased upon perfusing with K+-free medium. Increasing the temperature of perfusion medium from 25 to 40 degrees C or omitting glucose from the perfusing medium decreased the magnitude of oscillations. On the other hand, complete interruption of the perfusion flow resulted in an increase in the amplitude of pH oscillation. An initial increase followed by a decrease in the pH oscillation was seen when hearts were perfused with medium containing lactic acid at pH 6.6. These results suggest that pH oscillations reflect fluctuations in myocardial metabolism.     

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular Ca2+ transport in different areas of dog heart

Calcium transport and ATPase activities were determined in the heavy and mitochondrial fractions isolated from the left and right atria as well as ventricles of dogs. Ultrastructural distribution of these organelles in different areas of the myocardium was also examined. Calcium binding, calcium uptake, and calcium ATPase activities of the atrial microsomes were lower than those of the ventricles. On the other hand, mitochondrial calcium binding and uptake activities in the right atrium were higher than those in other areas. The mitochondrial total ATPase activities in the atria were also higher than those in the ventricles. Mitochondrial as well as microsomal yields from ventricles were significantly higher. Size and number of mitochondria in the ventricles were greater whereas no striking difference in the distribution of sarcoplasmic reticulum was apparent in different areas of the heart. Poorly developed calcium transport functions in the atrial microsomes may be one of the factors responsible for the generation of lower contractile force in this tissue in comparison with the ventricle.     

Protective effect of vanadate on oxyradical-induced changes in isolated perfused heart

In order to examine the mechanisms of the beneficial effects of vanadate on cardiac dysfunction in chronic diabetes, rat hearts were perfused with xanthine plus xanthine oxidase, an oxyradical generating system in the absence or presence of vanadate. The heart failed to generate contractile force and increased the resting tension markedly within 5 min of perfusion with xanthine plus xanthine oxidase. These changes were prevented by the addition of 4 M vanadate in the perfusion medium. The protective effects of vanadate on the loss of developed tension and increased resting tension due to xanthine plus xanthine oxidase were dose-dependent (0.1–5 M). Perfusion of the hearts with glucose-free medium did not abolish the protective actions of vanadate. The sarcolemmal Ca²⁺-pump (ATP-dependent Ca²⁺ uptake and Ca²⁺-stimulated ATPase) and Na⁺-dependent Ca²⁺ uptake activities were decreased upon perfusing the hearts with a medium containing xanthine plus xanthine oxidase for 5 min; these effects were prevented by the addition of 2–4 M vanadate in the perfusion medium. The signals for superoxide radicals produced by xanthine plus xanthine oxidase, as detected by electron paramagnetic resonance spectroscopic technique, were inhibited by 5–100 M vanadate. These results suggest that vanadate is an oxyradical scavenger and thus may prevent heart dysfunction under some pathological conditions by its antioxidant action.     

Relationship between mechanical dysfunction and depression of sarcolemmal Ca2+-pump activity in hearts perfused with oxygen free radicals

Although in vitro studies have shown that oxygen free radicals depress the sarcolemmal Ca²⁺-pump activity and thereby may cause the occurrence of intracellular Ca²⁺ overload for the genesis of contractile failure, the exact relationship between changes in sarcolemmal Ca²⁺-pump activity and cardiac function due to these radicals is not clear. In this study we examined the effects of oxygen radicals on sarcolemmal Ca²⁺ uptake and Ca²⁺-stimulated ATPase activities as well as contractile force development by employing isolated rat heart preparations. When hearts were perfused with medium containing xanthine plus xanthine oxidase, the sarcolemmal Ca²⁺-stimulated ATPase activity and ATP-dependent Ca²⁺ accumulation were depressed within 1 min whereas the developed contractile force, rate of contraction and rate of relaxation were increased at 1 min and decreased over 3–20 min of perfusion. The resting tension started increasing at 2 min of perfusion with xanthine plus xanthine oxidase. Catalase showed protective effects against these alterations in heart function and sarcolemmal Ca²⁺-pump activities upon perfusion with xanthine plus xanthine oxidase whereas superoxide dismutase did not exert such effects. The combination of catalase and superoxide dismutase did not produce greater effects in comparison to catalase alone. These results are consistent with the view that the depression of heart sarcolemmal Ca²⁺ pump activities may result in myocardial dysfunction due to the formation of hydrogen peroxide and/or hydroxyl radicals upon perfusing the hearts with xanthine plus xanthine oxidase.     

Subcellular effects of some anesthetic agents on rat myocardium.

The effects of ether, chloroform, and halothane on calcium accumulation and ATPase activity of rat heart microsomes and mitochondria as well as on myofibrillar ATPase activity were investigated. Chloroform and halothane depressed microsomal and mitochondrial calcium uptake and binding in a parallel fashion. Ether decreased microsomal calcium binding and mitochondrial calcium uptake to varying degrees, while mitochondrial calcium binding was slightly enhanced. Whereas ether had no effect, chloroform depressed microsomal and mitochondrial total APTase activities and halothane decreased microsomsl ATPase and slightly stimulated mitochondrial total ATPase activities. Halothane was found to depress myofibrillar Mg2+-ATPase and ether was capable of decreasing myofibrillar Ca2+-ATPase. Chloroform was seen to inhibit both myofibrillar enzymes. These results suggest that the cardiodepressant actions of volatile anesthetic agents may be due to alterations in the calcium accumulating abilities of microsomal and mitochondrial membranes while direct myofibrillar effects may contribute to the depression seen with relatively higher concentrations of anesthetics.     

Changes in force and calcium sensitivity in the developing avian heart.

The aim of this study was to characterize the development of the contractile properties of intact and chemically skinned muscle from chicken heart and to compare these characteristics with those of developing mammalian heart reported by others. Small trabeculae were dissected from left ventricles of Arbor Acre chickens between embryonic day 7 and young adulthood (7 weeks post-hatching). At all ages, increasing extracellular calcium (0.45-3.6 mM) progressively increased twitch force of electrically stimulated trabeculae. Twitch force at 1.8 mM extracellular calcium, normalized to cross-sectional area, increased to a maximum at 1 day post-hatching, remained constant through 3 weeks post-hatching, but then decreased at 7 weeks post-hatching. The maximal calcium-activated force of trabeculae chemically skinned with Triton X-100 detergent increased to a maximum 2 days before the time of hatching and was not significantly changed up to 7 weeks post-hatching. Over the ages studied, average twitch force in 1.8 mM calcium was between 26 and 66% of maximal calcium-activated force after skinning, suggesting that the contractile apparatus is not fully activated during the twitch in normal Ringer. In skinned trabeculae, the calcium sensitivity of the contractile apparatus was higher in the embryo than in the young adult. These age-dependent changes in calcium sensitivity are correlated with isoform switching in troponin T. A decrease in pH from 7.0 to 6.5 decreased the calcium sensitivity of the contractile apparatus to a greater degree in skinned trabeculae from young adult hearts than in those from embryonic hearts. This change in susceptibility to acidosis is temporally associated with isoform switching in troponin I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carnitine stimulation of glucose oxidation in the fatty acid perfused isolated working rat heart.

The effects of L-carnitine on myocardial glycolysis, glucose oxidation, and palmitate oxidation were determined in isolated working rat hearts. Hearts were perfused under aerobic conditions with perfusate containing either 11 mM [2-3H/U-14C]glucose in the presence or absence of 1.2 mM palmitate or 11 mM glucose and 1.2 mM [1-14C]palmitate. Myocardial carnitine levels were elevated by perfusing hearts with 10 mM L-carnitine. A 60-min perfusion period resulted in significant increases in total myocardial carnitine from 4376 +/- 211 to 9496 +/- 473 nmol/g dry weight. Glycolysis (measured as 3H2O production) was unchanged in carnitine-treated hearts perfused in the absence of fatty acids (4418 +/- 300 versus 4547 +/- 600 nmol glucose/g dry weight.min). If 1.2 mM palmitate was present in the perfusate, glycolysis decreased almost 2-fold compared with hearts perfused in the absence of fatty acids. In carnitine-treated hearts this drop in glycolysis did not occur (glycolytic rates were 2911 +/- 231 to 4629 +/- 460 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively. Compared with control hearts, glucose oxidation rates (measured as 14CO2 production from [U-14C]glucose) were unaltered in carnitine-treated hearts perfused in the absence of fatty acids (1819 +/- 169 versus 2026 +/- 171 nmol glucose/g dry weight.min, respectively). In the presence of 1.2 mM palmitate, glucose oxidation decreased dramatically in control hearts (11-fold). In carnitine-treated hearts, however, glucose oxidation was significantly greater than control hearts under these conditions (158 +/- 21 to 454 +/- 85 nmol glucose/g dry weight.min, in control and carnitine-treated hearts, respectively). Palmitate oxidation rates (measured as 14CO2 production from [1-14C]palmitate) decreased in the carnitine-treated hearts from 728 +/- 61 to 572 +/- 111 nmol palmitate/g dry weight.min. This probably occurred secondary to an increase in overall ATP production from glucose oxidation (from 5.4 to 14.5% of steady state myocardial ATP production). The results reported in this study provide direct evidence that carnitine can stimulate glucose oxidation in the intact fatty acid perfused heart. This probably occurs secondary to facilitating the intramitochondrial transfer of acetyl groups from acetyl-CoA to acetylcarnitine, thereby relieving inhibition of the pyruvate dehydrogenase complex.     

The role of calcium in the regulation of glucose uptake in isolated working rat heart

Catecholamines or ischemia may increase myocardial glucose uptake by an increase in intracellular calcium. We tested the hypothesis that increasing or decreasing extracellular calcium supply would change glucose uptake. Hearts were perfused for 60 min at a physiological workload with Krebs-Henseleit buffer containing glucose (5 mM) and oleate (0.4 mM; bound to 1% BSA). Calcium concentration was 2.5 mM. In group A (control; n = 12), insulin (1 mU/ml) was added at 30 min. In Group B (n = 7), the calcium concentration was increased to 5.0 and 7.5 mM at 20 min and 40 min, respectively. In Group C (n = 7), verapamil was added at 20 min (0.25 M) and 40 min (1.0 M) to decrease calcium influx. In group D (n = 7), EDTA was added at 20 min (0.5 mM) and at 40 min (1.5 mM) to decrease the free extracellular calcium. Glucose uptake was measured by ³H₂O production from [2-³H]glucose and cardiac work was measured simultaneously. Cardiac power in group B was 8.24 ± 0.60 mW at 2.5 mM calcium, 9.45 ± 0.50 mW at 5 mM calcium and 7.99 ± 0.99 mW at 7.5 mM calcium (n.s.). The addition of verapamil decreased contractile function in a dose-dependent manner (8.50 ± 0.74 vs. 3.11 ± 0.84 vs. 1.48 ± 0.39 mW, p < 0.01) suggesting that verapamil decreased cytosolic calcium concentration. A similar dose-dependent reduction in contractile performance was observed in the EDTA group (8.44 ± 0.81 vs. 7.42 ± 0.96 vs. 4.03 ± 1.32 mW, p < 0.01). Glucose uptake was 1.35 ± 0.11 mol/min/g dry weight under control conditions. Glucose uptake increased threefold with the addition of insulin. Increasing extracellular [Ca²⁺] did not affect glucose uptake. Decreasing Ca²⁺ availability showed a trend towards a decrease in glucose uptake (n.s.), which was minor compared to the decrease in contractile function. We conclude that extracellular calcium does not regulate glucose uptake in the isolated working rat heart in the presence of glucose and fatty acids as substrates. The trend of decreased glucose uptake when calcium supply was limited may be due to dramatically reduced energy demand and not directly due to changes in calcium.     

Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (;working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [(14)C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and (14)CO(2)). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue contents of hexose phosphates and of citrate. There were similar findings when working hearts from streptozotocin-diabetic rats with insulin added to the medium were compared with normal hearts. 8. The effects of insulin addition or of the chronic diabetic state could be explained in terms of an action of insulin on glucose transport. Increased heart work also acted at this site, but in addition there was evidence for altered regulation of glycolysis mediated by changes in tissue contents of adenine nucleotides or of citrate.     

Accumulation of amino acids in muscle of perfused rat heart. Effect of insulin

1. Rat heart perfused with Krebs-Henseleit bicarbonate buffer released material containing ninhydrin-positive nitrogen, but the amount was less than that reported to be released by diaphragm; glucose, but not insulin, decreased the release of ninhydrin-positive nitrogen and increased the concentration of the same material in the intracellular water of heart. 2. When heart was perfused with a mixture of amino acids and glucose, there was actually a net uptake, and an increase in intracellular concentration, of ninhydrin-positive nitrogen. Changes in the concentration of ninhydrin-positive nitrogen did not accurately reflect changes in concentration of amino acids. 3. The effect of insulin on the actual concentration of individual amino acids in heart muscle was examined by perfusing the heart with a mixture of amino acids and other ninhydrin-positive substances in the same concentration as they are found in plasma. 4. The effect of insulin on the concentrations of amino acids in the medium and in the intracellular water of the heart was determined after perfusion for different periods of time. No clear or meaningful effect of insulin was observed, despite the fact that insulin significantly increased the accumulation, in each of the same hearts, of radioactivity from amino[(14)C]isobutyric acid.     

Effects of lanthanum on the heart sarcolemmal ATPase and calcium binding activities.

Effects of lanthanum on Ca2+-ATPase, Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities were studied in rat heart sarcolemma. Ten to 100 micrometers lanthanum depressed significantly the Ca2+-ATPase activity and 50--200 micrometers lanthanum inhibited the calcium binding activity. Lineweaver-Burk plots of the Ca2+-ATPase activity showed that the inhibition by lanthanum was competitive with calcium concentration. Neither Mg2+-ATPase nor Na+-K+-ATPase activities were affected by lanthanum when the assay medium contained 1 mM EDTA; however, in the absence of EDTA, these enzyme activities were significantly decreased by 10--100 micrometers lanthanum. Rat hearts perfused with HEPES buffer containing 0.5 mM lanthanum showed electron-dense deposits restricted to the outer cell surface and the sarcolemma obtained from these hearts also had the deposits, indicating that the membrane fraction isolated by the hypotonic shock--LiBr treatment method is of sarcolemmal origin. The Ca2+-ATPase activity of the sarcolemma isolated from lanthanum-perfused hearts, unlike the Mg2+-ATPase, Na+-K+-ATPase, and calcium binding activities, was significantly less than the control value. From these observations it is suggested that lanthanum may influence calcium movement across the sarcolemma by affecting sarcolemmal ATPase and calcium binding activities.     

Intracellular glucose and binding of hexokinase and phosphofructokinase to particulate fractions increase under hypoxia in heart of the amazonian armored catfish (Liposarcus pardalis)

Armored catfish (Liposarcus pardalis), indigenous to the Amazon basin, have hearts that are extremely tolerant of oxygen limitation. Here we test the hypothesis that resistance to hypoxia is associated with increases in binding of selected glycolytic enzymes to subcellular fractions. Preparations of isolated ventricular sheets were subjected to 2 h of either oxygenated or hypoxic (via nitrogen gassing) treatment during which time the muscle was stimulated to contract. The bathing medium contained 5 mM glucose and was maintained at 25 degrees C. Initial experiments revealed increases in anaerobic metabolism. There was no measurable decrease in glycogen level; however, hypoxic treatment led to a twofold increase in heart glucose and a 10-fold increase in lactate content. It is suggested that the increase in heart glucose content is a result of an enhanced rate of facilitated glucose transport that exceeds the rate of phosphorylation of glucose. Further experiments assessed activities of metabolic enzymes in crude homogenates and subsequently tracked the degree of enzyme binding associated with subcellular fractions. Total maximal activities of glycolytic enzymes (hexokinase [HK], phosphofructokinase [PFK], aldolase, pyruvate kinase, lactate dehydrogenase), and a mitochondrial marker, citrate synthase, were not altered with the hypoxic treatment. A substantial portion (>/=50%) of HK is permanently bound to mitochondria, and this level increases under hypoxia. The amount of HK that is bound to the mitochondrial fraction is at least fourfold higher in hearts of L. pardalis than in rat hearts. Hypoxia also resulted in increased binding of PFK to a particulate fraction, and the degree of binding is higher in hypoxia-tolerant fish than in hypoxia-sensitive mammalian hearts. Such binding may be associated with increased glycolytic flux rates through modulation of enzyme-specific kinetics. The binding of HK and PFK occurs before any significant decrease in glycogen level.     

Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts

To study the mechanisms of mitochondrial dysfunction due to ischemia-reperfusion (I/R) injury, rat hearts were subjected to 20 or 30 min of global ischemia followed by 30 min of reperfusion. After recording both left ventricular developed pressure (LVDP) and end-diastolic pressure (LVEDP) to monitor the status of cardiac performance, mitochondria from these hearts were isolated to determine respiratory and oxidative phosphorylation activities. Although hearts subjected to 20 min of ischemia failed to generate LVDP and showed a marked increase in LVEDP, no changes in mitochondrial respiration and phosphorylation were observed. Reperfusion of 20-min ischemic hearts depressed mitochondrial function significantly but recovered LVDP completely and lowered the elevated LVEDP. On the other hand, depressed LVDP and elevated LVEDP in 30-min ischemic hearts were associated with depressions in both mitochondrial respiration and oxidative phosphorylation. Reperfusion of 30-min ischemic hearts elevated LVEDP, attenuated LVDP, and decreased mitochondrial state 3 and uncoupled respiration, respiratory control index, ADP-to-O ratio, as well as oxidative phosphorylation rate. Alterations of cardiac performance and mitochondrial function in I/R hearts were attenuated or prevented by pretreatment with oxyradical scavenging mixture (superoxide dismutase and catalase) or antioxidants [N-acetyl-L-cysteine or N-(2-mercaptopropionyl)-glycine]. Furthermore, alterations in cardiac performance and mitochondrial function due to I/R were simulated by an oxyradical-generating system (xanthine plus xanthine oxidase) and an oxidant (H(2)O(2)) either upon perfusing the heart or upon incubation with mitochondria. These results support the view that oxidative stress plays an important role in inducing changes in cardiac performance and mitochondrial function due to I/R.     

The effect of lanthanum on the mechanical function of the isolated perfused rat heart at different extracellular calcium concentrations.

The effects of 50 microM lanthanum (La3+) on the contractile force, rate and coronary flow of rat hearts perfused with solutions containing 2.5, 5, 7.5 mM calcium (Ca2+) have been investigated. La3+ produced a rapid and marked decrease in contractile force within 1-3 min ("early La(3+)-effect"). The inhibition of contractility by La3+ was reduced progressively when the Ca2+ ion concentration in the perfusion fluid was raised from 2.5 to 7.5 mM. However, after 10-80 min of La3+ perfusion the contractile force was increased significantly ("late La(3+)-effect"). Elevation of Ca2+ during exposure to La3+ increased its effect. During the late La(3+)-effect, a marked decrease in heart rate and a significant increase in time to reach peak tension, time for half relaxation and twitch duration was observed. High concentrations of perfusate Ca2+ decreased the chronotropic response to La3+, in contrast, elevated Ca2+ potentiated La(3+)-induced increase in time to reach peak tension, time for half relaxation and twitch duration. La3+ produced a significant decrease in coronary flow. High Ca2+ augmented the decrease coronary flow. The findings indicate that La3+ may produce marked effects on myocardial function. High extracellular Ca2+ reduces the La(3+)-induced initial decrease in force of contraction, but potentiates the late increase in contractile force by La3+. Elevated external Ca2+ also increases the effects of La3+ on twitch parameters, heart rate and coronary flow.     

Effect of prostacyclin on perfusion pressure, electrical activity, rate and force of contraction in isolated rat and rabbit hearts.

Prostacyclin when added to medium perfusing rat and rabbit hearts caused an increase in perfusion pressure at concentrations from 1 pg/ml ? 1 ng/ml (2.8 × 10^?12 ? 2.8 × 10^?9M) and a decrease at higher concentrations. Rhythm disturbances were observed with low prostacyclin concentrations in 6 of 10 rat hearts and 2 of 5 rabbit hearts studied. Increased heart rates were seen in the isolated rat hearts but not in the rabbit hearts. Force of contraction of isolated rat hearts was increased with increasing prostacyclin concentrations up to 100 pg/ml. Higher concentrations decreased contractile force. No inotropic effects were seen with rabbit hearts.     

Calcium incorporation by smooth muscle microsomes.

The purpose of the present work was to study the factors influencing calcium incorporation into a microsomal fraction prepared from the longitudinal smooth muscle of the guinea-pig ileum. Calcium incorporation required the presence of both ATP and Mg2+ and was unaffected by azide. It was enhanced by oxalate; this effect was pH dependent and it was maximal at pH 6.6. The relation between calcium uptake with oxalate and free Ca2+ concentration in the medium was represented by a curve with an optimum for Ca2+ equal to 3-10-5 M. The threshold concentration was comprised between 5-10-7 and 10-6 7. The optimum calcium uptake rate was 4.5 nmol Ca2+/mg protein per min. In the absence of oxalate, two distinct groups of binding sites were identified. Low affinity sites had a binding constant of 7-104 M-1 and a maximum binding capacity of 0.6-106 M-1 and a binding capacity of 33 nmol Ca2+/mg protein; their capacity was sensitive to pH changes. In the absence of oxalate, Ca2+ binding was depressed by Na+ with respect to K+ or choline. When the medium was supplemented with oxalate, the stimulation of 45Ca incorporation was barely detectable in the presence of choline+ and it was lower in a medium containing Na+ instead of K+. The subcellular distribution profiles of calcium incorporation with and without oxalate indicate the microsomal location of both activities. However, the oxalate-stimulated calcium uptake activity sedimented faster than the calcium binding activity. The subcellular distribution of marker enzyme actvities has been examined. The present results indicate that Ca2+ incorporations with and without oxalate are the result of two processes likely related to two different structures. The role of microsomal calcium uptake in excitation-contraction coupling and its modification by the activity of the sodium pump is discussed.     

Free fatty acids, but not ketone bodies, protect diabetic rat hearts during low-flow ischemia

To determine whether the effects of fatty acids on the diabetic heart during ischemia involve altered glycolytic ATP and proton production, we measured energetics and intracellular pH (pH(i)) by using (31)P NMR spectroscopy plus [2-(3)H]glucose uptake in isolated rat hearts. Hearts from 7-wk streptozotocin diabetic and control rats, perfused with buffer containing 11 mM glucose, with or without 1.2 mM palmitate or the ketone bodies, 4 mM beta-hydroxybutyrate plus 1 mM acetoacetate, were subjected to 32 min of low-flow (0.3 ml x g wet wt(-1) x min(-1)) ischemia, followed by 32 min of reperfusion. In control rat hearts, neither palmitate nor ketone bodies altered the recovery of contractile function. Diabetic rat hearts perfused with glucose alone or with ketone bodies, had functional recoveries 50% lower than those of the control hearts, but palmitate restored recovery to control levels. In a parallel group with the functional recoveries, palmitate prevented the 54% faster loss of ATP in the diabetic, glucose-perfused rat hearts during ischemia, but had no effect on the rate of ATP depletion in control hearts. Palmitate decreased total glucose uptake in control rat hearts during low-flow ischemia, from 106 +/- 17 to 52 +/- 12 micromol/g wet wt, but did not alter the total glucose uptake in the diabetic rat hearts, which was 42 +/- 5 micromol/g wet wt. Recovery of contractile function was unrelated to pH(i) during ischemia; the glucose-perfused control and palmitate-perfused diabetic hearts had end-ischemic pH(i) values that were significantly different at 6.36 +/- 0.04 and 6.60 +/- 0.02, respectively, but had similar functional recoveries, whereas the glucose-perfused diabetic hearts had significantly lower functional recoveries, but their pH(i) was 6.49 +/- 0.04. We conclude that fatty acids, but not ketone bodies, protect the diabetic heart by decreasing ATP depletion, with neither having detrimental effects on the normal rat heart during low-flow ischemia.