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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

The hormonal regulation and enzymatic basis of endogenous lipolysis in heart are not yet completely elucidated. The lysosomal fraction from rat heart appeared to be markedly enriched in triglycerides and a significant reduction in triglycerides in this fraction was found after prolonged perfusion or stimulation of lipolysis with glucagon. The enhanced rate of lipolysis, measured as glycerol release from the isolated perfused rat heart, was abolished 10-15 min after continuous glucagon administration. Omission of glucagon for another 60 min restored the ability of glucagon to stimulate lipolysis, indicating the limited availability of endogenous triglycerides and the presence of a transfer-system for triglycerides from a non-metabolically active pool to a metabolically active pool. The enhanced lipolysis induced by low-flow ischemia was found to be inhibited by the lysosomotropic agent methylamine (5 mM). Methylamine-perfusion during low-flow ischemia was accompanied by an increased recovery of myocardial triglycerides in the lysosomal fraction. The possible role of lysosome-like particles in myocardial triglyceride homeostasis was further investigated by studying the kinetics of uptake and degradation of labeled triglycerides by membrane-particles recovered in the subcellular fraction enriched with lysosomal marker enzymes. It appeared that isolated lysosomal membranes take up added triglycerides at an average rate of 30 nmoles/min/g protein. The bulk of these triglycerides taken up is stored whereas 20% is degraded to diglycerides and free fatty acids. More than 90% of the free fatty acids formed were released from the lysosomes into the supernatant. The uptake and degradation of triglyceride-filled liposomes by isolated myocardial lysosomes was inhibited during incubation with methylamine (5 mM). On the other hand, a lowering of pH during in vitro incubation increased the rate of uptake and degradation of added triglycerides by isolated lysosomes. These results indicate that lysosomes or lysosome-like particles are involved in the enhanced lipolysis during myocardial ischemia.

When the pH of the perfusion medium of rat Langendorff heart, paced at a rate of 300 beats/min, is abruptly lowered from pH 7.5 to 7.0, the hearts stop beating within 6 min in more than half of the cases. Reperfusion with pH 7.5 medium after 10 min pH 7.0 perfusion does not cause contractility to resume within 5 min. The causative factor is intracellular acidosis, resulting in severe morphological alterations of plasma membrane and mitochondria. It is probably initiated by the loss of membrane-bound calcium. Oleate, complexed with albumin included in the perfusion media, protects the hearts. This may be explained by maintenance of capillary flow and limitation of cellular acidosis.

Purified mackerel-oil extract or lard fat (9.1% wt/wt) was added to a basal diet of young pigs for 8 wk. Effects on plasma lipids, glucose and insulin, cardiac membrane phospholipids, cardiovascular performance, and morphology were studied. A time-dependent reduction of plasma triglyceride (62%), total cholesterol (41%), and HDL cholesterol (47%) was found in the mackerel-oil-fed pigs. The postprandial glucose and insulin response may indicate a mackerel-oil-induced resistance of insulin receptors. Although the polyunsaturated fatty acid composition of cardiac sarcolemma widely differed between the two groups, all determined indices of heart function were equal. It is concluded that consumption of a fish-oil diet with a low content of monoenes and supplemented with antioxidants reduces plasma lipid levels without producing pathological side effects.

The effects of feeding saturated and eucaloric unsaturated fat-rich diets on lipemia and insulinemia in female Brown Norway rats have been compared. The relative hyperlipemia in the unsaturated fat group at 8 a.m. has declined at 10 a.m., whereas the saturated fat group at 8 a.m. gives lower values than at 10 a.m. It suggests that saturated fat feeding requires a longer absorption period. The insulin levels in the unsaturated fat groups are higher at 8 a.m. than at 10 a.m., whereas insulin levels in the saturated fat group are higher at 10 a.m. than at 8 a.m. It also suggests retarded resorption of food in the saturated fat group. The relative hyperlipemia at 10 a.m. in the saturated fat group applies to triacylglycerol, free cholesterol, free fatty acids and particularly phosphatidylcholine. It was almost 50% higher than phosphatidylcholine in the unsaturated fat group and coincided with the accumulation of an apolipoprotein E-rich high density lipoprotein.

1. Albumin is the preferred stabilizer of the higher monoacylglycerol substrates, since the highest activity was measured with albumin rather than with Triton X-100 or other detergents tested. 2. The monoacylglycerol hydrolase activity may be strongly influenced by the amount of albumin used as the only emulsifier. Possible models for the physical states of the substrate are discussed. 3. The reaction rates with 1- and 2-monoacylglycerols are generally similar but may vary according to the physical states of the substrates. 4. The same enzyme hydrolyzes both 1- and 2-isomers since the hydrolytic activities were found to be competitive rather than additive. For both isomers identical apparent Km values less than 0.1 mM were obtained. 5. A comparison of the rates of hydrolysis of 1- and 2-monopalmitoylglycerol by the villus preparation at various temperatures confirmed that generally the reaction rates are similar and that the energy of activation is about 15 kcal/mol, so that the Q10 is about 1.8. 6. It is speculated that the microsomal level of long-chain acyl-CoA is an important determinant in the fate of the resorbed monoacylglycerol, since acylCoA is not only a substrate for the reacylation reactions but also an inhibitor of monoacylglycerol hydrolase.

Rat hearts were perfused for long periods in the presence of 14C-labeled amino acids. From these hearts, postheparin-effluent and a tissue homogenate containing lipoprotein lipase and neutral lipase, respectively, were derived. Lipolytic activity and 14C-labeled protein in both preparations were characterized by affinity chromatography, immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Lipase activity and 14C-labeled protein co-eluted from heparin-Sepharose 4B at 1.2 M NaCl and were inhibited and precipitated by preincubation with anti-lipoprotein lipase gamma-globulins. Gel electrophoresis of both preparations showed the presence of 14C-labeled protein with a molecular weight of 35 000. These data strongly suggest similarity between lipoprotein lipase and neutral lipase and their possible precursor-product relationship and indicate that during perfusion continuous synthesis, secretion and vascular binding of lipase molecules occur. Cycloheximide perfusion induced a dramatic decrease of lipoprotein lipase and neutral lipase activity, indicating a half-life of less than 90 min for both enzymes. Tunicamycin present during perfusion also induced a drop in lipoprotein lipase and tissue neutral lipase activity, indicating that glycosylation is necessary for secretion of lipoprotein lipase. Long-term perfusion of rat hearts in the presence of norepinephrine, glucagon or tyrosine leads to reciprocal alterations in lipoprotein lipase and neutral lipase activities, i.e., lipoprotein lipase activity increased and neutral lipase activity decreased, whereas total lipase activity (lipoprotein lipase + neutral lipase) remained unaltered. During perfusion in the presence of insulin, no net change in lipase activities was observed. Also, insulin did not affect the glucagon-induced inverse effects on either lipase activity. The reciprocal changes in lipase activities occurring during norepinephrine perfusion were hampered by colchicine and propranolol, pointing towards beta-receptor and microtubular mediation of tissue lipase processing and endothelial binding. Our data suggest that the tissue flux and vascular binding of lipase protein may be important sites of hormonal regulation of lipoprotein lipase homeostasis.

The alkaline, heparin-releasable lipoprotein lipase (LPL) activity of isolated, perfused rat hearts was compared with the residual neutral lipase (NL) activity detectable in the post nuclear supernatant (PNS) from a tissue homogenate. Both enzyme activities were increased by serum, heparin and apolipoprotein CII, inhibited by high salt concentrations and by immunotitration with an anti-LPL gamma-globulin fraction. Protamine sulphate from saline liver inhibited LPL activity and the NL activity only in the absence of serum. Incubation of the PNS NL under classic conditions of hormonal stimulation (by phosphorylation) did not alter its activity and upon short-term preperfusion of the hearts with norepinephrine and glucagon also unchanged LPL and NL activities were measured. Our experiments are indicative of a possible similarity between vascular LPL and tissue NL and show that the lipase activities are not sensitive towards hormonal stimulation.

The activity of liver lipase, an enzyme that can be released from the liver by heparin, varies under several hormonal conditions. The site(s) at which regulation of the enzyme activity may occur was investigated in vitro. As a model, rats were used which had been treated with a corticotrophin analogue, to induce hypercortisolism, a condition in which liver lipase activity is lowered. Lipases isolated from heparin-containing perfusates of livers from ACTH or control rats were identical with respect to heat stability and specific activity as determined by immunotitration and binding to isolated non-parenchymal liver cells, indicating that the enzyme structure was not affected by the treatment. The secretion of liver lipase by isolated parenchymal liver cells was studied. During incubation of parenchymal cells derived from ACTH rats, less enzyme activity was found to be secreted when compared with hepatocytes isolated from control rats (ACTH rats, 2.30 +/- 0.2 mU/10(6) cells; control rats, 3.3 +/- 0.3 mU/10(6) cells). Liver lipase partially purified from control rats could be bound specifically to saturation by non-parenchymal cells, isolated from ACTH or control rats. Non-parenchymal cells from ACTH rats bound less lipase activity (29 mU/mg cell protein) than cells from control rats (50 mU/mg cell protein). This reduction in binding capacity seems to be due to a diminished number of binding sites, since the affinity based on Scatchard analysis and half-maximal binding was not different. These results suggest that the lowered liver lipase activity found during hypercortisolism may be due to an impaired synthesis and/or secretion of the enzyme by the parenchymal cells and to a reduced binding capacity of the non-parenchymal cells for liver lipase.

The coronary flow rate of retrogradely perfused hearts from fasted (group I) and streptozotocin-diabetic rats (group II) is increased when compared with the flow rate of control, fed animals (group III). The enhanced coronary flow is absent when hearts of groups I and II are perfused in the presence of indomethacin (1 microgram/ml) in the perfusion fluid and the lowest flow rates are observed after depletion of the endogenous catecholamines by reserpin. Hearts from groups I and II showed a marked increase in prostaglandin-release which was counteracted both in the presence of indomethacin (1 microgram/ml) and by reserpin-pretreatment. The results suggest that the increased coronary flow rates in hearts from fasted and streptozotocin-diabetic rats are mediated by an effect of released endogenous catecholamines on coronary vascular smooth muscle and by a catecholamine-induced release of vasodilatory, prostaglandin-like substances.

The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprotein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a 10-times higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.

An overview is provided of current thinking regarding the origin(s) of atherosclerosis on the basis of clinical evidence, experimental models and pharmacological investigations with calcium antagonists. Because this group of drugs inhibits the influx of Ca2+ into the smooth muscle cell of the cardiovascular wall, it has been argued that when endothelial damage is anticipated or has occurred, the 'protective' action of calcium antagonists might avoid, delay or even help to repair the calcium overload seen with cell membrane damage. In the real world the situation must be far more complex as will become evident but as a concept the avoidance of Ca2+ overloading is enticing. In fact, based on circumstantial evidence, a major clinical trial is currently in progress in which the long term effect of nifedipine on coronary atherosclerosis is being evaluated in a double blind randomized fashion. The end-point of this study lies in the reduction of the severity and number of coronary artery lesions. Although not yet completed, the design and goals of that study are provided.

In intestinal epithelial cells, three enzymes possessing monoacylglycerol hydrolase activity were found and partially purified. Two of these enzymes have properties that justify their classification as an esterase and one as a monoacylglycerol lipase. The three enzymes show similar Km values for monooleoylglycerol and each shows similar activity towards 1- and 2-monopalmitoylglycerol. Antiserum raised in rabbits against rat liver monoacylglycerol lipase inhibits the intestinal lipase completely, suggesting that the enzymes are at least partially similar. The esterases of small intestinal villus cells were not inhibited by the antiserum against liver monoacylglycerol lipase. It was calculated that the esterases account for approx. 2/3 of the monooleoylglycerol hydrolase activity in epithelial cells. Monoacylglycerol lipase also hydrolyzed palmitoyl-CoA, while the esterases did not. The enzymes were inhibited by micellar palmitoyl-CoA. The hypothesis that palmitoyl-CoA is an important regulator for monoacylglycerol acylation is discussed in the light of these new findings.

1. A technique is described for the rapid separation of intestinal epithelial cells from the incubation medium by passage through a silicon-oil layer and collection in acid, in which their soluble constituents are released. 2. The inhibition by fatty acids of pyruvate oxidation is further studied. Measurement of pyruvate transport in epithelial cells at 0 degree C showed that short- and medium-chain fatty acids as well as ricinoleate inhibit this transport. Propionate inhibits pyruvate transport by another mechanism than octanoate. 3. Differences between pyruvate propionate and octanoate transport across the epithelial cell membrane were obtained in efflux studies. These studies revealed that acetate, propionate, butyrate and high concentrations of bicarbonate readily stimulate the efflux of pyruvate, probably by anionic counter-transport. No effects were seen with octanoate and hexanoate. The data obtained in these efflux studies suggest that lipophilicity and the pKa values of the monocarboxylic acids determine the contribution of non-ionic diffusion to overall transport. 4. Saturation kinetics, competitive inhibition by short-chain fatty acids and counter-transport suggest a carrier-mediated transport of pyruvate.

To determine the site of reperfusion damage after ischaemia the leakage of xanthine dehydrogenase and xanthine oxidase was assessed in vascular and interstitial effluents. Contractile function was reduced during hypoperfusion but improved after the addition of superoxide dismutase and vasoxin to the perfusion medium. Both interstitial fluid and coronary effluent showed dehydrogenase and oxidase activity after no flow ischaemia. Furthermore, the ratio of lactate dehydrogenase to creatine kinase in coronary effluents was reduced. These findings indicate that the myocardial interstitium may be a site of ischaemic membrane damage since this space contains hypoxanthine and xanthine oxidase. The protective effect of superoxide dismustase also indicates the possibility of damage due to oxygen derived radicals in the cardiac interstitium during low flow perfusion.

Hormonal and metabolic regulation of endogenous triglyceride hydrolysis was studied in triglyceride-enriched hearts obtained from rats fed 3 days with a trierucate-rich diet. Endogenous lipolysis was determined by measuring glycerol release during in vitro perfusion of the hearts. It appeared that there was a direct relation between the contractile state of the heart, the rate of glycerol release in the coronary effluent and the Ca2+ concentration in the perfusion medium. During Ca2+-free perfusion, 2,4-dinitrophenol stimulated oleate oxidation and this, as well as the addition of 2 x 10(-7) M glucagon, induced a marked stimulation of lipolysis. Insulin did not affect glucagon- and norepinphrine-stimulated lipolysis during substrate-free perfusion. The presented experiments point out that in lipid-enriched rat hearts the activity of the tissue lipase may be controlled by the rate of beta-oxidation and re-esterification of the liberated fatty acid, as well as by a shift to utilization of carbohydrate instead of fatty acids for energy supply.

1. In various preparations of rat small intestinal epithelial cells the acylation and deacylation of monoacylglycerol were studied. In the in vitro vascularly perfused intestine, of which the lumen was loaded with monoacylglycerol with or without fatty acids, acylation exceeded deacylation. In contrast, deacylation was much faster in isolated microsomes and in isolated whole cells. 2. In vascularly perfused intestine, without long-chain fatty acids present in the lumen, the amount of di- and triacylglycerol formed was found to be half of that formed in perfusions with long-chain fatty acids in the lumen, while the glycerol formation was increased 1.4-fold. 3. The concentration of monoacylglycerol is an important factor in determining the relative rates of monoacylglycerol acylation and deacylation in microsomes: the ratio acylation/deacylation decreases with increasing monoacylglycerol concentrations. 4. The function of the monoacylglycerol lipase in fat resorption is discussed.

Rat hearts were perfused retrogradely using a modified technique that allows the separate collection of coronary (Qrv) and interstitial (Qi) effluents. Evidence is presented that Qrv contains products from the coronary vasculature and that Qi contains products arising from cardiac myocytes. Heparin perfusion of rat hearts led to a release of lipolytic activity in Qrv and Qi which was characterized as lipoprotein lipase (LPL). The relative amounts of LPL released in Qrv and Qi were dependent on the feeding condition of the rat. A high LPL activity was recovered from Qrv of fasted rats, and Qi was high in LPL during feeding. On perfusion of hearts with [3H]cholesterol-labeled chylomicrons, the tissue uptake of cholesterol was highest in the fasted state, whereas release of radioactivity in Qi was predominant in the fed state. This radioactivity in Qi appeared to be associated with chylomicron degradation products (remnants and surface fragments). Our experiments indicate that cholesterol uptake during chylomicron breakdown is inhibited in the fed state, and the relationship between myocyte LPL activity and interstitial formation of chylomicron degradation products suggests a role for the myocyte LPL in lipoprotein metabolism.

1. The enzymes involved in glycerolphosphate and monoacylglycerol acylation of rat small intestine were more active in villi than in crypts. Monoglyceride acyltransferase (EC 2.3.1.22) was found to be absent from crypts. 2. In the villi, the enzymes are mainly localized in microsomes, although low activities of palmitoyl-CoA synthetase (EC 6.2.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15) and cholinephosphotransferase (EC 2.7.8.2) are found in mitochondria. Mitochondria lack monoglyceride acyltransferase and lysolecithin acyltransferase (EC 2.3.1.23), both of which are involved in the reacylation of alimentary partial glycerides. Therefore, this process is confined to microsomes. 3. The monoacylglycerol and lysolecithin acyltransferases, as well as choline-phosphotransferase, are probably localized within the endoplasmic reticulum, since these enzymes are relatively Nagerse resistant (subtilisin; EC 3.4.2.1, compared with palmitoyl-CoA synthetase and glycerolphosphate acyltransferase, which are highly Nagarse-sensitive and therefore probably localized on the outside of the microsomes (and mitochondria). 4. The physical separation of alimentary product reacylation from de novo synthetic processes provides the basis of metabolic compartmentation observed by other workers. 5. The use of sucrose instead of a salt medium for the isolation and homogenization of small intestinal epithelial cells allowed the separation of mitochondria and microsomes by differential centrifugation without mutual contamination. 6. Phospholipids were found to stimulate glycerolphosphate acylation in vitro. 7. The glycerolphosphate and monoacylglycerol acylation pathways are not competitive.