Release of ischemia in paced rat Langendorff hearts by supply of L-carnitine: role of endogenous long-chain acylcarnitine.

Rat Langendorff hearts perfused with media that do not contain erythrocytes or fluorocarbon as oxygen carriers are borderline aerobic during 5 Hz pacing. This follows from the release of catabolic products measured: lactate, urate and Iysophosphatidyl-choline (IysoPC). Addition of L-carnitine to the perfusion medium reduced the level of these compounds, while the release of long-chain acylcarnitine (LCAC) increased. Previously, we found (Biochim Biophys Acta 847:62-66,1985) that micromolar LCAC protects membranes during reperfusion after ischemia. Therefore, the observed inverse relation between LCAC and the other compounds measured suggests that LCAC is the basis of an acute relief of imminent ischemia by carnitine addition. LCAC may be released from various cell types, including vascular endothelium, as demonstrated. The cationic amphiphilic nature of LCAC is responsible for protection of membrane functions in imminent ischemia.

Quaternary nitrogen compounds affect carnitine distribution in rats. Particular emphasis on edrophonium.

Edrophonium (ethyl(m-hydroxyphenyl)dimethylamine) acutely modifies carnitine levels in different rat tissues, increasing hepatic and reducing blood and renal levels. After 2 h edrophonium treatment, the total serum carnitine levels were decreased by 16 (P < 0.001) and 33 (P < 0.001) percent in fed and fasted rats respectively compared to control, and in kidney the levels decreased by 11 (P < 0.05) and 34 (P < 0.001) percent whereas in liver the edrophonium treatment increased the levels by 43 (P < 0.001) and 59 (P < 0.001) percent. The edrophonium action does not depend on the route of administration or on the nutritional state of the animal. Its activity on carnitine levels is neither accompanied by significant variation of serum parameters of carbohydrate, fat and protein metabolism nor of insulin levels. The edrophonium activity is not related to cholinergic action, as physostigmine and ambenonium at concentrations known to increase cholinergic activity do not modify carnitine distribution in tissues. Trimethylphenylammonium (TPA) and trimethyl(p-aminophenyl)ammonium (TPA.NH2), compounds structurally similar to edrophonium, are on the contrary active on levels of carnitine and this effect is not related to their cholinergic potency. In 24 h fasted rats after the TPA and TPA. NH2 treatment, the total serum carnitine levels were decreased by 32 (P < 0.001) and 13 (n.s.) percent respectively compared to control, and in kidney the levels decreased by 15 (P < 0.02) and 5 (n.s.) percent, whereas in liver the treatment increased the levels by 72 (P < 0.001) and 45 (P < 0.01) percent. Moreover atropine, an acetylcholine antagonist, affects carnitine distribution in a way similar to edrophonium. Edrophonium activity on carnitine distribution, probably affects (inter)cellular carnitine transport by direct action on plasma membrane. Effect on capillary endothelium may be responsible for its observed action on muscle contraction force in imminent ischemia.

Uptake and release of carnitine by vascular endothelium in culture; effects of protons and oxygen free radicals.

The present paper shows that cultured bovine endothelial cells can be labeled with 3H-carnitine by incubation. This process is slow and is uphill, requiring Na+/K+ ATPase activity. After 3 days incubation isotopic equilibrium is reached, when the cells contain about 0.5 mM (total) carnitine at a medium concentration of about 3 microM. The plasmamembrane barrier is rather resistant to acidosis and oxygen free radicals (OFR). The rate of carnitine release increases significantly only at pH below 5.8. At pH 6.0 the release of stored carnitine can be initiated by the addition of D- or L-lactate. OFR, generated by the addition of xanthine and xanthine oxidase, did not affect carnitine release. Both mild acidosis and OFR left plasmamembranes of endothelial cells intact as judged by the absence of lactate dehydrogenase loss from the cells. Therefore, the known increase of capillary permeability during ischemia and reperfusion may not be due to plasmalemmal disruption of individual endothelial cells, but to increase of inter-endothelial spaces.

Alteration of tissue carnitine content following anaesthesia with barbiturate and surgery in rat.

The hypercatabolic state leads to urinary carnitine loss. Anaesthesia and surgical intervention causes stress conditions. The stress is accompanied by the alteration of hormone states and energy processes. Carnitine, which physiologically promotes the fatty acid metabolism and so plays a very important role for the heart, can be involved in these alterations. The aim of this study was to examine the effects of anaesthesia and surgical intervention on carnitine distribution in the tissues. Rats were anaesthetized with thiopental and surgical intervention was performed on the femoral artery and vein. Carnitine levels, as well as parameters indicative of energy metabolism, were measured in the blood and tissues. It was found that anaesthesia and surgical intervention increased the corticosterone levels in blood and decreased the carnitine levels in blood and kidney. Carnitine accumulated in the liver, whereas in heart and skeletal muscle it was redistributed by a decrease in the acylated form and an increase in the free form. Glycogen was accumulated in cardiac and skeletal muscle. Compared to anaesthesia, the surgical intervention increased glycogen storage and carnitine redistribution in heart and skeletal muscle. Moreover, it caused a decrease in cholesterol and an increase in urea in the blood. The fall in blood and kidney carnitine levels indicates a possible depletion of carnitine. The deacylation of carnitine in heart and skeletal muscle represents an important alteration in heart and muscle energetic metabolism. The increase in urea is a consequence of high proteolysis. Acylcarnitine administration during pre- and operative step might prevent the loss of carnitine, promoting the heart energetic metabolism and reducing the proteolysis. Moreover, in accordance with a recent interpretation of carnitine action as a membrane-stabilizing agent, the acylcarnitine supply could reduce the risk of "oedema" that follows the anaesthesia and surgical intervention.

Comparison of the effects of carnitine palmitoyltransferase-1 and -2 inhibitors on rat heart hypertrophy.

Rats treated orally for 21 days with aminocarnitine, an inhibitor of carnitine palmitoyltransferase-2 (CPT-2), do not show hypertrophy of the heart. This contrasts with the effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, that, according to the literature, cause hypertrophy. As CPT-1 and CPT-2 are both required for the oxidation of long-chain fatty acids in mitochondria, it can be concluded that inhibition of fatty acid oxidation per se is not responsible for cell growth, but rather the accumulation of a metabolite, probably long-chain acylcoenzyme A. CPT-1 and CPT-2 inhibitors cause different metabolic changes in the heart. Electron microscopy of hearts fixed 1 hour after Langendorff perfusion with the two types of inhibitors reveals some of these changes. Multilamellar vesicles were observed with aminocarnitine (CPT-2 inhibitor) but not with etomoxir (CPT-1 inhibitor). When both inhibitors were present, electron-dense spots adjacent to mitochondria were observed, possibly containing long-chain acylaminocarnitine.

Carnitine and cardiac interstitium.

An important part of (acyl)carnitine may be stored in interstitial spaces and the external surface of adjacent cells. A high concentration of carnitine in the direct vicinity of cells may enhance the synthesis and export of long-chain acylcarnitine. Long-chain acylcoenzyme A, from which long-chain acyl carnitine is formed, cannot penetrate intact cell membranes. During hypoperfusion or ischemia, when long-chain acylcoenzyme A accumulates due to hampered fatty acid oxidation, there is an increased formation of long-chain acyl carnitine which diffuses into the interstitium and adjacent vascular endothelial cells. Due to its lipophilic nature and net positive charge (limitation of carboxyl-group dissociation in ischemic acidosis), long-chain acyl carnitine may decrease the affinity of Ca2+ to the cell surface and prevent Ca2+ overload of cells. The advantage of carnitine over many other cationic amphiphiles in the protection of areas of ischemia is that long-chain acyl carnitine is formed and stored only in ischemic areas.

L-carnitine combined with minimal electrical stimulation promotes type transformation of canine latissimus dorsi.

In the first part of this study, in four dogs the left latissimus dorsi was equipped to perform in vivo contraction measurements and the right latissimus dorsi served as control. After a control period, the dogs received L-carnitine intravenously for 8 wk. We found that carnitine caused the percentage of type I fibers to increase from 30 to 55% in the left latissimus dorsi but no change in the right latissimus dorsi. In the left latissimus dorsi, the contraction speed (percentage ripple) decreased from 75 to 30% and cytochrome-c oxidase activity increased 1.6-fold. No changes occurred in the right latissimus dorsi. To verify these observations, we performed a second study with placebo control for 8 wk, and only the left latissimus dorsi was subjected to weekly electrical stimulation. In the carnitine-treated dogs, the stimulated muscle showed an increase in the percentage of type I fibers from 16 to 35% and the ripple decreased from 92 to 77%. These measures did not change in the placebo-treated dogs. We concluded that weekly short-term stimulation does not lead to a change in fiber type; however, carnitine combined with minimal stimulation of the muscle leads to a significant shift in muscle fiber type composition toward a muscle with an increased content of type I fibers.

Imminent ischemia in normal and hypertrophic Langendorff rat hearts; effects of fatty acids and superoxide dismutase monitored by NADH surface fluorescence.

Hypertrophic hearts contain areas of hypoperfusion which can be visualized by increased NADH surface fluorescence during in vitro perfusion without oxygen-carrying particles under constant pressure and pacing. By contrast, fluorescence remained low when non-hypertrophic hearts were used instead. When during perfusion of normal hearts the pH of the medium was lowered from 7.5 to 7.0, areas of high fluorescence appeared in a few minutes. The high fluorescent areas under conditions of cardiac hypertrophy or pH 7.0 perfusion could be reduced by addition of superoxide dismutase. It indicates that oxygen free radicals interfere with proper flow regulation in areas of low pH. Fluorescence in hypertrophic hearts also diminished during addition of albumin-bound oleate to the standard, glucose-containing, medium. This is in agreement with our earlier finding of fatty acid protection from acidosis-initiated loss of capillary flow (Biochim. Biophys. Acta, 1033 (1990) 214-218). In contrast to low concentrations of free fatty acids, high concentrations interfere with tissue oxygenation. This has been illustrated by the use of 1 mM octanoate, which after a few min caused the appearance of high fluorescent areas. We conclude that decompensation of flow in hypoperfused areas of heart, as occurs in hypertrophy, may be stimulated by acidosis and oxygen free radicals.

Vulnerability of vascular endothelium in lipopolysaccharide toxicity: effect of (acyl) carnitine on endothelial stability.

The literature presented illustrates that lipopolysaccharide (LPS), from bacterial cell walls, induces tumour necrosis factor (TNF) synthesis in macrophages. TNF affects a number of cell types, amongst which are endothelial cells, within a few hours. Its injection has been shown to produce all symptoms of the toxic syndrome. In the present communication the vulnerability of endothelial cells will be stressed. These cells require carnitine not only for fatty acid oxidation but also for membrane protection and repair. As endothelial cells lose carnitine during hypoperfusion, it is speculated that the supply of carnitine during the early phase of LPS toxicity in rats might delay or avoid loss of endothelial functions. Earlier it was observed that hearts from rats, injected 3 h previously with LPS, showed strongly increased interstitial fluid production compared to hearts from control rats, even when TNF was present during a 3 h in vitro perfusion. It showed that LPS in vivo generates factors other than TNF, such as platelet activating factor (PAF), that are responsible for the increased capillary permeability.

Carnitine requirement of vascular endothelial and smooth muscle cells in imminent ischemia.

Vascular endothelial and -smooth muscle cells have been shown to use fatty acids as substrates for oxidative phosphorylation. Endothelial cells are more vulnerable to oxidative stress than muscle cells and are prone to loose carnitine early during hypoperfusion. This has been suggested by two observations. The first is that incubation of isolated endothelial cells in a low carnitine medium leads to oleate oxidation, dependent upon carnitine addition, whereas smooth muscle cells do not depend on carnitine addition during in vitro incubation, although aminocarnitine, a specific inner-membrane carnitine palmitoyltransferase inhibitor, inhibits fatty acid oxidation. The second observation is that rat hearts labeled in vivo with 14C-carnitine loose, as paced Langendorff heart, only 4% of their carnitine in 20 min perfusion, following 60 min global ischemia. The carnitine released had a much higher specific radioactivity than the carnitine that was not released. It indicates compartmentation of carnitine in heart. As earlier and presently discussed work shows endothelial vulnerability, it is to be expected that this cell type may become carnitine deficient during pacing and ischemia. Endothelial incompetence in flow regulation could be delayed by the presence of carnitine and fatty acids in pre-ischemia. It is speculated how activated fatty acids could protect endothelium.

Accumulation and excretion of long-chain acylcarnitine by rat hearts; studies with aminocarnitine.

During Langendorff perfusion of rat heart with aminocarnitine, long-chain acylcarnitine (LCAC) accumulates in heart cells, from which it is excreted by the heart. The heart function remains intact during this process. The accumulation of LCAC can be inhibited by the simultaneous addition of an inhibitor of the outer membrane carnitine palmitoyl-coenzyme A transferase (CPT-1), indicating that aminocarnitine is a specific inhibitor of the inner membrane isoenzyme (CPT-2). LCAC accumulation is associated with glycogen depletion. After 60 min perfusion with aminocarnitine, electron microscopy shows large multilamellar lipid vesicles, especially in cardiomyocytes, which are depleted in glycogen granula. Multilamellar lipid vesicles are also found in the blood vessels. Extraction of the perfusate shows the presence of LCAC, fatty acid and phosphatidylethanolamine. Morphological analysis with freeze fracturing and thin sectioning furthermore reveals that the sarcolemma is not deteriorated during the export of LCAC to the coronary vessels. Since cardiac structures and functions are intact, LCAC alone is not the clue for ischemic damage. Therefore the present work supports the hypothesis that acidosis rather than LCAC is of primary importance to ischemic damage.

The effect of L-carnitine on force development of the latissimus dorsi muscle in dogs.

Using the latissimus dorsi (LD) muscle of the dog in situ, the effect of carnitine was tested for increase of force in the first period after stimulation. Carnitine administration resulted in an increase of force of 31 +/- 6% (mean +/- SEM). It is hypothesized that, during muscle stimulation, a relative carnitine deficiency occurs in cells of the vascular compartment. The previously observed lesser effect of carnitine in the trained muscle than in the untrained muscle is in line with this hypothesis, since the number of capillaries is known to increase by training. Also in agreement with this hypothesis is the observation that carnitine increased flow during exercise of the muscle.

Acute effect of L-carnitine on skeletal muscle force tests in dogs.

Using the mixed type musculus latissimus dorsi of the dog in the present work, we show the effect of carnitine on an in situ fatigue test. L-Carnitine appears to improve force of this muscle by 34% while stimulated in situ. This effect of carnitine is acute and (stereo)specific, since neither D-carnitine nor the structural analogue choline (also a tertiary amine) has a positive effect on contractile force. Because skeletal muscle is rich in carnitine and because carnitine transport is slow, its effect must be exerted outside the striated muscle cells. Insulin (with glucose) administration abolished the carnitine effect. It is speculated that facilitation of fatty acid oxidation in the blood vessel wall is the basis for this positive effect of carnitine.

Biochemical profile of propionyl-L-carnitine.

This article briefly discusses biochemical reactions involved in the metabolism of propionate, propionyl-CoA, and propionyl-L-carnitine in the heart. The aim is to understand the way in which propionyl-L-carnitine can exert a protective effect on the ischemic/reperfused heart. The protection of the plasmalemma by propionyl-L-carnitine during acidosis of the heart is also discussed. One protective mechanism is based on the ability of propionate to replenish mitochondria with dicarboxylic acid intermediates of the citric acid cycle and to increase the cellular content of carnitine, both of which may stimulate the generation of energy in the postischemic reperfusion phase. Another mechanism presumes a stabilizing action of acylcarnitines upon biomembranes.

Hormonal control of cardiac lipolysis by glyco(geno)lysis.

The importance of the glucose/fatty acid cycle in the control of cardiac lipolysis is emphasized by the following observations. Addition of the glycogen debranching inhibitor deoxynojirimycin or an O2-vehicle, fluorocarbon F-43, to media perfusing paced, lipid-enriched, Langendorff hearts lower cardiac lactate and glycerol 3-phosphate levels together with inhibition of glucagon-stimulated glycerol (and lactate) release. The absence of fluorocarbon during perfusion of 5 Hz paced langendorff hearts probably results in limited tissue oxygenation, resulting in glycogenolysis and lipolysis. The results indicate hormonal control of cardiac lipolysis by glyco(geno)lysis.

Inhibition of carnitine palmitoyltransferase leads to induction of 3-hydroxymethylglutaryl coenzyme A reductase activity in rat liver.

The relation between carnitine palmitoyltransferase (CPT) activity and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated. Rats were treated with aminocarnitine or 1-carnitine overnight. In rats, in which CPT activity was inhibited by aminocarnitine, plasma and hepatic triacylglycerol contents were increased 5- to 6-fold. The plasma cholesterol concentration was unchanged, while the hepatic cholesterol content was lowered (-16%). Hepatic cholesterol synthesis, determined by following the incorporation of 14C-acetate and 3H2O into digitonin-precipitable sterols, in liver slices was increased 5- to 7-fold. HMG-CoA reductase activity in liver microsomes was increased to the same extent.

Properties of phosphatidate phosphohydrolase and diacylglycerol acyltransferase activities in the isolated rat heart. Effect of glucagon, ischaemia and diabetes.

Myocardial triacylglycerol hydrolysis is subject to product inhibition. After hydrolysis of endogenous triacylglycerols, the main proportion of the liberated fatty acids is re-esterified to triacylglycerol, indicating the importance of fatty acid re-esterification in the regulation of myocardial triacylglycerol homoeostasis. Therefore, we characterized phosphatidate phosphohydrolase (PAP) and diacylglycerol acyltransferase (DGAT) activities, enzymes catalysing the final steps in the re-esterification of fatty acids to triacylglycerols in the isolated rat heart. The PAP activity was mainly recovered in the microsomal and soluble cell fractions, with an apparent Km of 0.14 mM for both the microsomal and the soluble enzyme. PAP was stimulated by Mg2+ and oleic acid. Oleic acid, like a high concentration of KCl, stimulated the translocation of PAP activity from the soluble to the particulate (microsomal) fraction. Myocardial DGAT had an apparent Km of 3.8 microM and was predominantly recovered in the particulate (microsomal) fraction. Both enzyme activities were significantly increased after acute streptozotocin-induced diabetes, PAP from 15.6 +/- 1.1 to 28.1 +/- 3.6 m-units/g wet wt. (P less than 0.01) and DGAT from 2.23 +/- 0.11 to 3.01 +/- 0.11 m-units/g wet wt. (P less than 0.01). In contrast with diabetes, low-flow ischaemia during 30 min did not affect PAP and DGAT activity in rat hearts. Perfusion with glucagon (0.1 microM) during 30 min did not affect total PAP activity, but changed the subcellular distribution. More PAP activity was recovered in the particulate fraction. DGAT activity was lowered by glucagon treatment from 0.37 +/- 0.03 to 0.23 +/- 0.02 m-unit/mg of microsomal protein (P less than 0.05). The role of PAP and DGAT activity and PAP distribution in the myocardial glucose/fatty acid cycle is discussed.

Acidosis, cardiac stunning and its prevention by oxygen.

When during aerobic perfusion of the 5 Hz paced rat Langendorff heart, under constant aortic pressure of 8.3 kPa, the pH of the medium is changed from 7.5 to 7.0 a short period of positive inotropy is followed by a dramatic loss of contractility. The hearts, rapidly frozen after 10 min pH 7.0 perfusion, show moderate loss of high-energy phosphates and accumulation of lactate and glycerol-3-phosphate, indicative of tissue anaerobiosis. This can be overcome by including fluorocarbon, an O2 vehicle, in the media. The transient positive inotropy is interpreted as H(+)-induced release of plasmalemma-bound Ca2+ into the cytosol. The accompanying morphologic alterations are as described in this issue by Vandeplassche and Borgers (1990) and by Verkleij et al. (1990).