Calcium-free Perfusion Research Articles

Tissue protection by adrenergic blockade in the calcium paradox?

In a graded model of the calcium paradox phenomenon (minimal and total) the presence of the beta-blocker propranolol (5 X 10(-6) M) in the perfusion media (10 min prior to, during and 5 min following calcium-free perfusion) had no effect upon tissue injury. Propranolol pretreatment (three days prior to the experiments) significantly reduced the myocardial enzyme release during calcium repletion in the minimal calcium paradox. The presence of the alpha 1-blocker prazosin (1 X 10(-7) M) in the perfusion media (10 min prior to, during and 5 min following calcium-free perfusion) afforded no protective effects. It is concluded that the release of endogenous catecholamines may not be an important factor contributing to myocardial injury in the calcium paradox, and that consequently beta- or alpha 1-adrenergic blockade has little if any protective properties in this form of myocardial injury.

Calcium paradox-evoked release of prostacyclin and immunoreactive leukotriene C4 from rat and guinea-pig hearts. Evidence that endogenous prostaglandins inhibit leukotriene biosynthesis

The purpose of this study was to assess the influence of the calcium paradox (5 min calcium-free perfusion followed by 15 min calcium repletion) on the release of immunoreactive leukotriene C4 and 6 Keto-prostaglandin F1 alpha from rat and guinea-pig hearts. Under control conditions or during the 5 min calcium-free perfusion period no immunoreactive leukotriene C4 was detectable in the coronary effluent. Following reperfusion with calcium-containing medium a large release of leukotriene C4 was observed although the amount was significantly greater in the rat heart. 6 keto-prostaglandin F1 alpha was detected during normal and calcium-free perfusion and the release was significantly stimulated during calcium repletion. Treatment with ibuprofen, a cyclo-oxygenase inhibitor, prevented the release of 6 keto-prostaglandin F1 alpha but increased the efflux of immunoreactive LTC4 during calcium repletion. Arachidonic acid, the substrate for prostaglandin and leukotriene synthesis increased the efflux of 6 keto-prostaglandin F1 alpha but decreased the release of leukotriene C4. The latter effect was reversed by perfusion with ibuprofen, and mimicked by prostacyclin, the primary cardiac prostaglandin. This study shows that the calcium paradox is a potent stimulus for eicosanoid release from rat and guinea-pig hearts, a phenomenon likely due to the activation of calcium-dependent enzymes. The study also suggests that endogenous prostaglandins inhibit leukotriene synthesis in cardiac tissue.

31P NMR study of intracellular pH during the calcium paradox

Reperfusion of an isolated mammalian heart with a calcium-containing solution after a brief calcium-free perfusion results in irreversible cell damage: the calcium paradox. It has been suggested that acidification of the cytosol, as a result of hydrolysis of ATP and accumulation of calcium by mitochondria, is an important factor in the development of the calcium paradox. Phosphorus nuclear magnetic resonance (31P NMR) spectroscopy was used to investigate the course of intracellular pH during the calcium paradox in isolated rabbit heart at 37 degrees C. Intracellular pH was measured from the chemical shift of the intracellular inorganic phosphate (Pi) peak. During control perfusion and the subsequent calcium-free period intracellular pH amounted to 7.1. After induction of the calcium paradox by readmitting calcium to the perfusion fluid, intracellular pH amounted to 7.0. It is concluded that acidification of the cytosol does not play a causal role in the development of the calcium paradox.

Acetylcholine-induced contraction in human isolated bronchial smooth muscle: role of an intracellular calcium store

The aim of this study was to investigate the role played by an intracellular calcium store in human bronchoconstriction. Human isolated brochial smooth muscle strips (5–6 mm long, 0.4–0.5 mm wide) were taken from lung specimens during pneumonectomy. Isometric contraction was recorded after stimulation by 10 −4 M acetylcholine in physiological solutions. The amplitude of acetylcholine-induced contractions was measured in the presence and absence of calcium ions in the perfusing medium. When the perfusing medium was switched to a calcium-free solution the amplitude of the acetylcholine-induced contraction was measured with respect to the duration of calcium-free perfusion. The amplitude of the contraction was 82.1 ± 11.3%, 77.2 ± 15.4%, 3 ± 10.6% of the maximum contraction after teh strips were perfused in calcium-free solutions for 1, 3 and 5 min respectively. Several successive contractions could be elicited, and even after 20 min of calcium-free perfusion, acetylcholine was still able to elicit contractions. These results suggest that an intracellular calcium store may be involved in human bronchoconstriction. This finding may help further our understanding of the effects of calcium antagonists on human airways.

Free fatty acid metabolism in "stunned" myocardium.

To assess whether myocardial lipid metabolism is altered in the "stunned" myocardium we have studied the metabolism of (1-14C)-palmitate during reperfusion in a modified rat heart preparation. Hearts were perfused retrogradely at a physiological flow rate (2 ml/min) in a non-recirculating system with erythrocyte-enhanced Krebs-Henseleit buffer containing albumin 0.4 mM, glucose 11 mM, palmitate 0.4 mM and trace amounts of (1-14C)-palmitate. Left ventricular pressure was measured by a latex balloon in the left ventricular cavity. Control hearts were perfused at constant flow for 120 min. To achieve reversible ischaemic damage, myocardial perfusion was reduced by 95% for 40 min, followed by reperfusion at the control flow rate for 60 min (reperfusion group). For comparison, irreversible damage was produced by calcium free perfusion (calcium paradox group). In the reperfusion group, the developed pressure was severely depressed 5 min after reperfusion to 23% of the value in the control group (p less than 0.05) but recovered to 84% (NS) at 60 min. In the calcium paradox group, mechanical activity ceased completely without recovery. Myocardial uptake of (1-14C)-palmitate in the reperfusion group was similar to the control experiments for the entire reperfusion period, whereas a marked depression was observed in the calcium paradox group. 14CO2 production was severely depressed at the onset of reperfusion in both the reperfusion and calcium paradox group to 42% (p less than 0.05) and 29% (p less than 0.05) respectively. In contrast to the calcium paradox group, 14CO2 production in the reperfusion group recovered progressively to 70% (NS) of the control value during the 60 min of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial protection by micromolar manganese in the calcium paradox and additive effects of verapamil.

The aims of the present study were to establish if micromolar manganese (50 mumol) may substitute for calcium during calcium-free perfusion and also to assess if manganese substitution for calcium may ensure tissue protection by verapamil (1.0 mg/l). Two injury levels (minimal and total calcium paradox) caused by different volumes (5 ml and 45 ml) of calcium-free perfusion (5 min) prior to calcium repletion (15 min) were examined in a normothermic isolated rat heart model. The presence of manganese during calcium-free only (5 min) or the presence of manganese prior to (5 min), during (5 min), and following (5 min) calcium-free perfusion conferred considerable protection as assessed by enzymatic, physiological and metabolic parameters in both the total and minimal calcium paradox models. The presence of verapamil prior to (5 min), during (5 min) and following (5 min) calcium-free perfusion combined with the presence of manganese conferred a further protection, particularly in the total paradox. It is concluded from the study that 50 mumol of manganese, although effective in tissue protection, cannot substitute totally for the loss of calcium during calcium-free perfusion and that manganese may substitute for the micromolar calcium needed for tissue protection by verapamil.

Protection by verapamil in the calcium paradox: dependence on micromolar calcium.

Myocardial protection by verapamil in the calcium paradox has been the subject of considerable controversy. In order to reassess the protective properties of verapamil in the normothermic isolated rat heart three injury levels (minimal, subtotal, total calcium paradox) caused by increasing flow rates (1, 2, 9 ml . min-1) or volumes (5, 10, 45 ml) of calcium free perfusion (5 min) before calcium repletion (15 min) were determined in the presence and absence of verapamil (2 mumol . litre-1) using enzymatic, metabolic, and physiological indices. Verapamil supplementation had minimal effect on tissue injury in the 45 ml and 10 ml volume group of hearts but afforded considerable protection in the 5 ml volume group. The addition of EDTA (0.1 mmol . litre-1) to the calcium free perfusate in the 5 ml volume group produced a subtotal calcium paradox on which verapamil had no effect. Conversely, when 20 mumol . litre-1 calcium was added to the otherwise calcium free perfusate in the 45 ml volume group a minimal calcium paradox was created on which verapamil had a substantial protective effect. It is concluded that tissue protection by verapamil in the calcium paradox requires the presence in the myocardium of both verapamil and trace amounts of calcium before the onset of calcium repletion. Tissue protection by verapamil is therefore limited to minimal injury types.

Septal release of vasopressin in response to osmotic, hypovolemic and electrical stimulation in rats

The central release of vasopressin was studied in anesthetized rats using push-pull perfusions and and radiommunoassay of the hormone. A basal release was observed in the lateral septum and in the lateral ventricle, whereas no vasopressin was dected in the perfusates from the caudate nucleus. Under osmotic stimulation, vasopressin release increased up to 12 and 60 times basal levels following i.p. injections of 5 ml and 10 ml/kg b. wt. of 2 M NaCl, respectively. This increase was blocked by using a calcium-free perfusion medium containing 0.1 mM EGTA. In the lateral ventricle, osmotic stimulation (5 ml/kg of 2 M NaCl i.p.) had the same effect as in the septum. In the caudate nucleus, no release was observed. Hemorrhage also increased the septal of vasopressin in 5 out of 6 animals tested. Electrical stimulation of the pituitary stalk and of the supraoptic nucleus was used to evoke the release of vasopressin into the bloodstream. Septal release slightly decreased during pituitary stalk stimulation, whereas it did increase during stimulation of the supraoptic region. Our results show that systemic stimuli for vasopressin release evoke both a peripheral and a septal release of the hormone. The dissociation of the effects of electrical stimulation of the pituitary stalk and of the supraoptic nucleus suggests, however, that the vasopressinergic neurones responsible for septal release are distinct from those which project to the neurohypophysis.

Myocardial cAMP and calcium levels in the calcium paradox.

In a graded model (minimal, subtotal, total) of the calcium paradox phenomenon induced by a progressive increase in the flow rate and volume (5 ml, 10 ml, 45 ml) of calcium-free perfusion (5 min) the severity of tissue injury on calcium repletion was related to a potential elevation of cAMP during calcium depletion. In the subtotal and total models of the calcium paradox a 50% increase was found for tissue cAMP after calcium-free perfusion, but no such rise could be associated with the minimal calcium paradox. In the study tissue injury, as assessed by whole tissue and mitochondrial accumulation of calcium and by myocardial leakage of creatine kinase, could only partly be related to cAMP changes. It is concluded that calcium-free coronary perfusion induces a complex series of events favouring excessive calcium entry, only one of which may be related to a change in cAMP.

The effect of hypothermia during the period of calcium repletion on the calcium paradox

Reperfusion of an isolated heart with a calcium-containing solution after a short calcium-free perfusion may result in irreversible cell damage: the calcium paradox. In this investigation the effect of hypothermia during reperfusion with calcium-containing solution on the calcium paradox damage in the isolated rat heart was studied. In addition, the effect of pre-cooling the heart during the calcium-free period was investigated. Creatine kinase release was used to define cell damage. Normothermic (37 degrees C) calcium-free perfusion followed by normothermic reperfusion with calcium-containing solution resulted in a massive release of CK. When the normothermic calcium-free perfusion was followed by hypothermic (10 degrees C) calcium-containing reperfusion, CK release was reduced by 20% (P less than 0.005). This CK release during reperfusion was further reduced by 55% and 80% when the normothermic calcium-free perfusion was followed by 5 or 10 min respectively of hypothermic calcium-free perfusion prior to the hypothermic calcium-containing reperfusion. The results show that hypothermia during the period of calcium repletion retards the sequence of events which ultimately results in release of large amounts of intracellular components.

Mitochondrial and cytoplasmic aspartate aminotransferase enzyme release in the calcium paradox.

The release of mitochondrial and cytoplasmic aspartate aminotransferase (AST) enzymes from the myocardium was studied in the isolated rabbit heart under conditions of the calcium paradox. Four different periods of calcium-free perfusion for 10, 15, 20, and 25 min were selected to produce different degrees of the calcium paradox and the associated myocardial damage which was indicated by impairment in the left ventricular contractile function. Calcium-free perfusion periods of less than 20 min were associated with partial recovery of ventricular function, while periods of 20 min or greater were associated with little or no recovery of contraction after reperfusion with calcium. Mitochondrial (ASTm) and cytoplasmic (ASTc) aspartate aminotransferase were released from the heart beginning within 1 min of reintroduction of Ca2+. The cumulative amount of ASTm release was about one-tenth the amount of ASTc release. The cumulative amount of ASTm and ASTc released were significantly (p less than 0.05) related to the duration of calcium-free perfusion. The time to 90% of maximum AST release was slightly longer for ASTm compared with ASTc (6.8 +/- 0.6 vs. 5.7 +/- 0.5 min, 0.10 greater than p greater than 0.05). ASTc but not ASTm correlated significantly (p less than 0.05) with total protein release from the myocardium, while ASTm was not as consistently related to protein loss. The cumulative amount of ASTm and ASTc were inversely related to the extent of recovery of left ventricular contractile function. Disparities did occur as the longest duration of the calcium-free period, which did not produce any further damage to left ventricular function, was nonetheless associated with more enzyme release from the myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathways for the movements of ions during calcium-free perfusion and the induction of the ‘calcium paradox’

The intracellular sodium content of cardiac cells in fish and amphibia, measured with either an isotope technique or with sodium-sensitive micro-electrodes, rises steeply from around 15 mmol/l in calcium-containing solution to as much as 70 mmol/l, during exposure to a Ca2+-free solution. This increase is associated with the development of spontaneous and prolonged action potentials so that the membrane may stabilise around -20 mV. On reperfusion with calcium-containing medium the membrane repolarises before a strong contracture develops. Inhibition of the Na-pump increases both the sodium gain and the subsequent calcium re-admission tension. A number of agents e.g. divalent cations, anti-arrhythmic drugs, local anaesthetics and Ca-channel blockers are able to prevent the development of the contracture but only if they are present during the calcium-free perfusion. They also inhibit the development of spontaneous electrical activity and the rise in Nai. The calcium re-admission contracture can be blocked in amphibian preparations voltage clamped around the resting potential during low calcium perfusion. From the known pharmacological action of these agents and the voltage and time dependence of the calcium channel, it is concluded that during calcium depletion, the prolongation of the action potentials is associated with a sustained entry of Na+ via the Ca-channels which leads to the rise in Nai. Once Nai has risen, these agents with the exception of Mn2+, a known inhibitor of the Na/Ca exchange, are unable to prevent the development of the contracture. This suggests that the re-admission contracture follows calcium uptake by way of the Na/Ca exchange.

Myocardial norepinephrine and cyclic AMP content during calcium free perfusion

Myocardial norepinephrine and cyclic AMP content during calcium free perfusion

Release of unassembled rat cardiac myosin light chain 1 following the calcium paradox.

To determine the intracellular source and release kinetics of myosin light chain 1 immediately following irreversible myocytic injury, we perfused rat hearts in a Langendorff apparatus under control conditions (20 minutes), or during global cellular injury produced by oxygenated, calcium-free perfusion (5 minutes), followed by reperfusion with buffer containing 2.5 mM calcium (15 minutes). Light chain 1 concentration (double antibody radioimmunoassay) and creatine kinase activity were measured in both the coronary effluent and the 140,000 g supernatant extract of perfused ventricular tissue (after homogenization and ultracentrifugation). Calcium reperfusion caused the rapid release of both light chain 1 and creatine kinase activity (peak light chain 1 = 1.09 +/- 0.19 micrograms/g; peak creatine kinase = 74.9 +/- 10.7 IU/ g at 1 minute, mean +/- SD, n = 3); 28.5 +/- 13.5% of total light chain 1 and 86.5 +/- 0.6% of total creatine kinase activity were depleted from the tissue extract during the 15-minute reperfusion. No light chain 1 or creatine kinase was detected in the effluents of control-perfused hearts. Dodecyl sulfate polyacrylamide gel electrophoresis and immunodetection with specific antibody to myosin heavy chain and light chain 1 showed that the effluent light chain 1 was of similar molecular weight (mol wt = 27,000) to the subunit bound to myofibrils. In addition, light chain 1 was released in the absence of myosin heavy chain. Thus, a small soluble pool of unassembled myosin light chain 1 subunits exists in the cytoplasm of cardiac myocytes that is released from irreversibly injured cells. This pool demonstrates initial washout kinetics similar to creatine kinase.

Effect of calcium depletion and calcium paradox on myocardial energy metabolism.

Both phases of the calcium paradox were associated with major alterations in myocardial energy metabolism. During calcium-free perfusion contractility of the heart ceased, resulting in a dramatic decrease in anaerobic and aerobic metabolism but no change in tissue high energy phosphate levels. Tissue content of most citric acid cycle intermediates were elevated, while there was a net decrease in the content of transaminase-linked amino acids. Reperfusion of the calcium-depleted heart with calcium-containing buffer failed to restore either the contractile or the metabolic state of the heart. Within seconds following calcium repletion, tissue high energy phosphate content plummeted. This occurred even though glucose utilization increased significantly and aerobic metabolism remained at levels observed in the calcium-depleted heart. Analogous to changes seen in acidosis and ischemia, alpha-ketoglutarate and citrate levels decreased abruptly. After a short delay, the levels of several key amino acids also dropped. The results support the hypothesis that the impairment of mitochondrial function contributes to the depletion of high energy phosphate stores during the calcium paradox.

The calcium paradox phenomenon: A flow rate and volume response study of calcium-free perfusion

A dose-response study concerning the importance of the flow rate (0.5 to 12 ml/min) and volume (2.5 to 60 ml) of calcium-free coronary perfusion (duration 5 min) in the induction of a calcium paradox on reperfusion (duration 15 min) with calcium-containing medium has been performed in the isolated rat heart (37 degrees C). On the basis of enzymatic, physiological, and metabolic assessments three different levels of tissue injury were identified: a minimal paradox at 1.0 ml/min or 5 ml, a subtotal paradox at 2 ml/min or 10 ml and a total paradox at 9 ml/min or 45 ml. Ultrastructural examination revealed that cellular injury following calcium repletion was always severe, and that an increase in the flow rate and volume of calcium-free perfusion increased the number of severely injured cells. During calcium-free perfusion the external lamina largely remained intact over the surface coat of the sarcolemma, but variable degrees of separation of intercalated discs were observed. It is concluded that the calcium paradox model of myocardial injury presents a rather sharp threshold related to the flow rate or volume of calcium-free coronary perfusion and that on trespassing this threshold there is a narrow zone characterized by a decreasing number of viable cells. Furthermore, the study indicates that a separation of the external lamina from the surface coat of the sarcolemma is not a prerequisite for the induction of a calcium paradox, and that cell injury may occur in the presence of intact intercalated discs.

Myocardial protection utilizing calcium containing and calcium free perfusates.

The effects of the presence or absence of calcium in the cardioplegic perfusate were studied utilizing the isolated blood perfused dog heart preparation. Hearts were subjected to two hours of arrest at 27 degrees C followed by 90 minutes of normothermic reperfusion. Perfusates with or without 2.52 mM calcium chloride were delivered at 15 minute intervals during arrest at a perfusion pressure of 100 mm Hg. Both calcium and calcium free perfusates resulted in decreases in tissue calcium concentration measured at the end of arrest. Tissue magnesium concentrations did not change with either perfusate. Coronary vascular resistance was increased with early perfusions in the calcium perfused group relative to the calcium free group. Systolic and diastolic performance, high energy phosphate values, coronary blood flow and myocardial oxygen consumption were altered by the arrest-reperfusion sequence but no differences between calcium and calcium free groups were seen. Thus, no evidence for deleterious effects of calcium free perfusion was present after two hours of global cardiac arrest followed by reperfusion. The data indicate the absence of the calcium paradox under conditions simulating clinical cardioplegia and supports clinical data indicating that the addition of calcium to the cardioplegic solution is unnecessary.

Ultrastructural studies of intercalated disc separations in the rat heart during the calcium paradox.

The ultrastructure of intercalated disc separations were studied in isolated rat hearts subjected to 5 min of coronary perfusion with small volumes of a calcium-free solution (i.e., 10.0 ml, 5.0 ml, and 2.5 ml). The same groups of hearts were studied after 15 min of calcium repletion. A semiquantitative examination shows that after calcium depletion 20%-45% of the intercalated discs (ID) were separated in the 2.5-ml group, 50%-75% in the 5.0-ml group, and 75%-90% in the 10.0-ml group. Readmission of calcium did not give any significant changes in the percentage of ID dehiscence in the two lowest volume groups, which indicates that ID separation has been irreversible during the first 15 min of calcium repletion. A semiquantitative analysis has also been performed of the percentages of severely damaged cells at each of the three volume groups after calcium repletion. It appears that in the two lowest volume groups, the percentage of widened discs tend to exceed the percentage of severely injured cells after calcium readmission. This suggests that ID separation not necessarily implies severe injuries to the implicated cells during calcium repletion. After calcium-free perfusion, cellular edema, cytoplasmic disintegration, and plasmalemmal fragmentation were present in the interdigitating cellular projections of the dissociated ID. Similar injuries did also occasionally occur outside the ID, usually situated in close proximity to a capillary.

Sarcolemmal disruption during the calcium paradox

Reperfusion of an isolated heart with calcium-containing solution after a short period of calcium-free perfusion may result in irreversible cell damage (calcium paradox). The ultrastructure of the sarcolemma of the rabbit heart during the calcium paradox was studied by using fast freezing devices. This method excluded ultrastructural changes induced by chemical fixation and cryoprotection. In addition, thin-section and conventional freeze-fracture electron microscopy were used. During reperfusion with calcium-containing solution disruption of the sarcolemma was observed, which was attended with formation of unilamellar and multilamellar vesicles and aggregation of intramembrane particles. These ultrastructural changes are explained in terms of calcium- and proton-induced lateral phase separation and fusion processes in the lipid bilayer of the sarcolemma.

Myocardial protection by micromolar manganese in the calcium paradox and additive effects of verapamil

The aims of the present study were to establish if micromolar manganese (50 mumol) may substitute for calcium during calcium-free perfusion and also to assess if manganese substitution for calcium may ensure tissue protection by verapamil (1.0 mg/l). Two injury levels (minimal and total calcium paradox) caused by different volumes (5 ml and 45 ml) of calcium-free perfusion (5 min) prior to calcium repletion (15 min) were examined in a normothermic isolated rat heart model. The presence of manganese during calcium-free only (5 min) or the presence of manganese prior to (5 min), during (5 min), and following (5 min) calcium-free perfusion conferred considerable protection as assessed by enzymatic, physiological and metabolic parameters in both the total and minimal calcium paradox models. The presence of verapamil prior to (5 min), during (5 min) and following (5 min) calcium-free perfusion combined with the presence of manganese conferred a further protection, particularly in the total paradox. It is concluded from the study that 50 mumol of manganese, although effective in tissue protection, cannot substitute totally for the loss of calcium during calcium-free perfusion and that manganese may substitute for the micromolar calcium needed for tissue protection by verapamil.