Interaction of carnitine with mitochondrial cardiolipin.

The physiological role of L-carnitine is to determine the transport of acyl-CoA through the mitochondrial membrane. However, some observations may also suggest a direct effect of the molecule per se on the physical properties of the membrane, most probably at the level of the binding site. This possibility has been investigated by studying the influence of adriamycin, a drug that binds to cardiolipin, on the effect of carnitine on isolated rat liver mitochondria. It has been found that adriamycin almost abolishes the activating effect of carnitine on state 2 respiration. The effect and its inhibition is seen by using either the L-form of carnitine or the D-form or both. Cardiolipin removes the effect of adriamycin and restores the activation by carnitine. It is proposed that some effects of carnitine on mitochondrial properties may be the result of interaction of carnitine with cardiolipin at the membrane level.

Effects of L-carnitine and its acetate and propionate esters on the molecular dynamics of human erythrocyte membrane.

EPR and fluorescence probes were used in this study to define the effects of L-carnitine and its short-chain esters, acetyl-L-carnitine and propionyl-L-carnitine, on the natural fluidity gradient and molecular packing of phospholipid headgroups of erythrocyte membrane in intact cells. Purified erythrocyte suspensions, labeled with different stearic acid derivatives containing a stable doxyl radical ring at the C-5, C-7, C-12 and C-16, were incubated with 0.5-5 mM L-carnitine and its esters for 60 min at 37 degrees C and washed twice with an isosmotic buffer. A decrease in the order parameter, calculated from the EPR spectra of the 5-doxylstearic acid derivative, was observed at all the concentrations of propionyl-L-carnitine and the extent of the decrease was dose and temperature dependent. An increase of the chain length between the doxyl ring and the carboxylic group of the spin label, resulted in a much lower efficacy of propionyl-L-carnitine in decreasing the order parameter. Acetyl-L-carnitine also showed a significant effect of decreasing the molecular order but only at the lower temperatures of red cells labeled with 5-doxyl and treated with the highest concentration of the drug. L-Carnitine did not modify the molecular dynamics at all the temperatures and concentrations used in this study. L-Carnitine and its short-chain derivatives did not alter significantly membrane fluidity of deeper regions of the erythrocyte membrane, measured by means of the excimer/monomer fluorescence intensity ratio of pyrene incorporated into the membrane of intact erythrocytes. However, these compounds were all capable of loosening the molecular packing of the polar head of erythrocyte membrane phospholipids evaluated by the membrane binding fluorescence properties of merocyanine-540. The binding of the fluorescent probe decreased in the order propionyl-L-carnitine > acetyl-L-carnitine > L-carnitine. Our findings suggest that this category of compounds affect the molecular dynamics of a membrane bilayer region close to the glycerol backbone of phospholipids, which might be relevant for the expression of membrane functions.

Sites of action of carnitine and its derivatives on the cardiovascular system: interactions with membranes.

Carnitine plays an essential role in the regulation of long-chain fatty acid metabolism in skeletal and cardiac muscle, a process that is mediated by well-characterized enzymatic mechanisms. Here, Irving Fritz and Edoardo Arrigoni-Martelli review the evidence that carnitine and its O-acyl derivatives also influence membrane fluidity, ion channel functions, smooth muscle contractility, membrane stability and cardiac functions. The authors present the view that direct interactions of carnitine derivatives with cell membranes are independent of reactions catalysed by carnitine acyltransferases. They propose that the novel actions discussed are implicated in the mechanisms by which carnitine and its derivatives protect perfused hearts subjected to ischaemia or to oxidative stress, and help people suffering from certain types of myocardial ischaemia or peripheral arterial disease.

Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L-carnitine.

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4-6 weeks duration were that 24-h urinary carnitine excretion was increased approximately twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10-15%) and sciatic nerve changes in Na(+)-K(+)-ATPase activity (decreased 50%), Mg(2+)-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.

The protecting effect of L-carnitine on Ca(2+)-loaded rat liver mitochondria.

It is shown that L-carnitine strongly increases the ability of rat liver mitochondria to respond to the train of Ca2+ additions by a transient stimulation of the State-4 respiration rate. Such an effect requires ATP and the L-carnitine efficiency strongly decreases when ATP is omitted. Oleate influences the mitochondria in a fashion opposite to that of L-carnitine. The oleate effect is strongly diminished by L-carnitine. Again, the L-carnitine effect requires ATP, and D-carnitine fails to substitute for L-carnitine. It is suggested that L-carnitine removes, in an ATP-dependent manner, endogenous or added fatty acids, which are involved in oxidative damage of Ca(2+)-loaded mitochondria.

Uncoupling effect of fatty acids on heart muscle mitochondria and submitochondrial particles.

The effect of ATP/ADP-antiporter inhibitors on palmitate-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitate is found to decrease the respiration-generated membrane potential. In mitochondria, this effect is specifically abolished by carboxyatractylate (CAtr) a non-penetrating inhibitor of antiporter. In submitochondrial particles, CAtr does not abolish the palmitate-induced potential decrease. At the same time, bongkrekic acid, a penetrating inhibitor of the antiporter, suppresses the palmitate effect on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in mitochondria as well as in particles decreases the palmitate uncoupling efficiency in both these systems. These data are in agreement with the hypothesis that the ATP/ADP-antiporter is involved in the action of free fatty acids as natural uncouplers of oxidative phosphorylation.

Synthesis and hypotensive activity of N-alkyl-N"-cyano-N'-pyridylguanidines.

A variety of N-alkyl-N'-pyridyl-N"-cyanoguanidines III was prepared as potential bioisosteres of hypotensive N-alkyl-N'-pyridylthioureas Ia. Optimal activity of the N,N'-disubstituted cyanoguanidines III was assoicated with the presence of four to seven carbon branched alkyl and 3- or 4-pyridyl groups. Maximum potency was displayed by N-tert-pentyl-N'-3 pyridyl-N"-cyanoguanidine (20). This compound proved to be 200 times more potent than the corresponding thiourea in hypertensive rats and dogs. In comparison with guancydine, which is the de-3-pyridyl analogue of 20, a 150-fold increase of potency in spontaneously hypertensive rats was obtained with 20 and its tert-butyl analogue 19. The observed activity appears to be due to direct vascular relaxation. On a weight basis compounds 19, 20, 50, and 101 compared favorably with hydralazine.

Basic antiinflammatory compounds. N,N',N''-Trisubstituted guanidines.

A variety of basic N,N',N'',-trisubstituted guanidines was prepared and tested for antiinflammatory activity. Compounds with a thiazolylguanidine moiety linked to the 4 position of the 2-methylquinoline ring exhibited fairly high antiinflammatory activity. Optimal activity was associated with the presence of N-cycloalkyl substituents on N''-4-(2-methylquinolyl)-N'-2-thiazolylguanidine. Pharmacological data on N-cyclohexyl-N''-4-(2-methylquinolyl)-N'-2-thiazolylguanidine (SR 1368, 44) are presented and discussed.

Multiple effects of a new anti-inflammatory agent, timegadine, on arachidonic acid release and metabolism in neutrophils and platelets.

Casein-elicited rat peritoneal polymorphonuclear leukocytes (PMNL) and rabbit platelets were prelabelled with [1-14C]arachidonic acid, and the effect of timegadine, a new anti-inflammatory agent, on the release and metabolism of arachidonic acid induced by A23187 (PMNL) and thrombin (platelets) was studied and compared with the effect of other compounds reported to affect these enzymatic mechanisms. Timegadine inhibited arachidonic acid release from both cells (IC50 = 2.7 X 10(-5) M), the lipoxygenase activity in PMNL (IC50 = 4.1 X 10(-5) M) and the cyclooxygenase activity in platelets (IC50 = 3.1 X 10(-8) M). By these mechanisms, PMNL leukotriene B4 formation was inhibited by 50% at 2.0 X 10(-5) M, platelet thromboxane B2 at 3.2 X 10(-8) M, and platelet 12-HETE at 4.9 X 10(-5) M. These effects might add to the understanding of the anti-inflammatory properties of timegadine.

L-carnitine uptake into primary rat cortical cultures: interaction with GABA.

The ability of the primary rat cortical cells to take up L-carnitine increased with the age of the cultures and plateaued at around day 11 up to 25 days in vitro (DIV) when a slight decline was evident and by 32 DIV there was a major decrease in L-carnitine uptake. The uptake of L-carnitine displayed complex components. Elimination of mitochondrial energy supply by NaCN (1 mM), rotenone (1.25 microM) and DNP (50 microM), caused a small but significant decrease in the uptake (21, 11 and 16%, respectively). The uptake was highly dependent on the Na gradient, since ouabain (0.5 mM) and Na free buffer (replaced by 250 mM sucrose), reduced uptake by 54 and 63%, respectively. There was competition of L-carnitine uptake by molecules resembling its structure, e.g. gamma-aminobutyric acid (GABA), acetyl-L-carnitine (ALC), D-carnitine, L-aminocarnitine and L-choline, with GABA being the most potent inhibitor (57% at 50 microM) and L-choline not being significantly active. The Na-dependent uptake of L-carnitine was saturable with a high Km (692 microM) and Vmax (839 pmol/min/mg). This Na-dependent component was not further additive with the GABA (500 microM) or the DNP (50 microM) inhibitable component, suggesting that it represented the same phenomenon, probably the Na gradient dependent transport of L-carnitine. The results indicate that the uptake of L-carnitine occurs by Na-dependent saturable process as well as non-saturable, Na-independent processes. At least the former uptake mechanism is potently inhibited by GABA.

Culture of dorsal root ganglion neurons from aged rats: effects of acetyl-L-carnitine and NGF.

In vitro neuronal preparations are used to study the action mechanism of substances which are active in normal and pathological brain aging. One major concern with in vitro assays is that the use of embryonic or adult neurons may hamper an appreciation of the relevance of these substances on aged nervous tissue. In the present study for the first time cultures of aged dorsal root ganglia from 24-months-old rats were maintained in vitro up to 2 weeks. This model was used to investigate the neurotrophic/neuroprotective action of nerve growth factor and acetyl-L-carnitine. A large population of aged dorsal root ganglia neurons was responsive to nerve growth factor (100 ng/ml). Nerve growth factor induced an increase of initial rate of axonal regeneration and influenced the survival time of these neurons. Acetyl-L-carnitine (250 microM) did not affect the axonal regeneration but substantially attenuated the rate of neuronal mortality. A significant difference was evident between the acetyl-L-carnitine-treated and the untreated neurons from the first cell counting (day 3 in culture). After 2 weeks the number of aged neurons treated with acetyl-L-carnitine was almost double that of the controls. The effects of acetyl-L-carnitine on aged DRG neurons potentially explain the positive effects in clinical and in vivo experimental studies.

Effects of acetyl-L-carnitine on the survival of adult rat sensory neurons in primary cultures.

Acetyl-L-carnitine produces a significant increase in the survival time-course of adult rat sensory neurons maintained in primary cultures up to 40 days. The analysis of our data suggests that 200 microM acetyl-L-carnitine added to the medium, slows down neuronal decay especially in the first 10 days in vitro, sparing a fraction of cells which would otherwise be lost. Patch-clamp recordings from these neurons show that superfusion with acetyl-L-carnitine (100-1000 microM) does not induce any membrane current. In addition an agonist muscarinic effect particularly concerning high-voltage activated calcium channel modulation appears to be ruled out. In conclusion our data favour the role of acetyl-L-carnitine in the trophism of sensory neurons in adult rats. In agreement with other in vivo experiments our data reinforce the hypothesis that this substance might be involved in reducing neuronal loss observed in nervous system aging.

Vascular effects in dogs of pinacidil (P 1134), a novel vasoactive antihypertensive agent.

In pentobarbital sodium-anaesthetized dogs, pinacidil was infused for approximately 5 min into the carotid, coronary, femoral or renal artery at a rate of 10 micrograms/kg per min. The infusion, which did not affect systemic blood pressure, rapidly and markedly increased blood flow to any of the regions studied. When given i.v., 0.2 mg/kg pinacidil caused a moderate reduction in mean arterial blood pressure (15-20 mmHg) associated with an increase in coronary and renal blood flow while femoral and carotid blood flow remained unchanged; 0.5 mg/kg led to a marked (40-60 mmHg) reduction in blood pressure associated with an increase in coronary blood flow whereas renal, carotid and femoral blood flow stabilized at control levels. Indomethacin (2.5 mg/kg i.v.) failed to reverse the hypotension induced by pinacidil. The results are in accord with the concept that the vascular effect of pinacidil is due to direct smooth muscle relaxation which does not depend on prostaglandin synthesis.

The effects of kininase II inhibition by SQ 14225 on kidney kallikrein-kinin and prostaglandin systems in conscious dogs.

The novel kininase II inhibitor SQ 14225 was intravenously administered to normal conscious dogs at the dose of 0.1 mg/kg. Urine kinin excretion increased from 14 +/- 6 ng/h to 163 +/- 18 ng/h. Separation by column chromatography showed that the increased urine kinin excretion was due to increased excretion of bradykinin. The enhanced urinary kinin excretion was associated by increased sodium (70%) and decreased kallikrein (27%) excretion. Urine volume and urinary prostaglandin excretion were not significantly affected by SQ 14225 treatment.

Positive action of propionyl-L-carnitine on mechanical performance of papillary muscle from Syrian hamsters with hereditary dilated cardiomyopathy.

Propionyl-L-carnitine has been shown to exert a beneficial effect on cardiac function in different experimental models of cardiomyopathy in the rat, most likely by improving cardiac metabolism and energy production. We have previously shown that, in a strain of hamsters with hereditary dilated cardiomyopathy (BIO TO.2), the mechanical activity of papillary muscle (length-tension, velocity of shortening, shortening, work and power relationship) is significantly depressed when compared to the same parameter in normal hamsters (BIO F1.B). The repeated oral treatment with propionyl-L-carnitine (60 mg/kg per os for 7 weeks) to BIO TO.2 hamsters had a significant positive inotropic effect, as indicated by an increase in developed tension up to the levels observed in papillary muscles from normal hamsters. This action is most likely associated with metabolic effects similar to those observed in rats.

Endothelins urinary excretion is increased in spontaneously diabetic rats: BB/BB.

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.

Partial cerebral ischemia assessed by "in vivo" 31P NMR spectroscopy in rats.

31P NMR spectroscopy was used to assess the cerebral ischemia status in rats by measuring the relative levels of phosphate metabolites. Partial cerebral ischemia was induced in 49 rats by reversible occlusion of the carotid arteries. Rats were intubated and mechanically ventilated on a hypoxic gas mixture. Physiological parameters such as temperature and arterial pressure were strictly controlled during the experiments. 31P spectra were acquired at 7 T during basal observation, for 15-20 min after the induction of ischemia, and for 1 hr after reperfusion. Depletion and increase in PCr and Pi levels, respectively, were already observable in the collected spectra within few minutes after the onset of ischemia. No appreciable changes were found in the ATP levels.

Pharmacokinetics of 2-(alpha-thenoylthio)-propionylglycine (TTPG) in healthy volunteers--an oral dose-proportionality investigation.

The dose linearity of 2-(alpha-thenoylthio)-propionylglycine (TTPG) pharmacokinetics after a single oral administration at three different TTPG doses (180, 540 and 1080 mg) was evaluated in 12 healthy volunteers according to an open, randomized, cross-over study with a 1-week wash-out period between each administration. The duration of the study, for each subject, was 4 weeks. Plasma concentration and urinary excretion of TTPG and its two systemic metabolites, namely propionylglycine (tiopronin) and thiophenecarboxylic acid (TCA) were assayed by a previously well validated HPLC method. Due to differences in the physical and chemical properties of these compounds, two assays were needed, one to measure TTPG and TCA as such, and one to measure derivatized tiopronin. Both used UV detection. TTPG, tiopronin and TCA were quickly detected in plasma, suggesting that the drug administered is rapidly absorbed and biotransformed, in part, in the systemic circulation into the two metabolites noted above. Time-to-peak for all three analytes showed a trend to increase with increasing doses of TTPG, being: 0.42, 0.40 and 0.67 h (P < 0.01) with TTPG; 0.53, 0.47 and 0.73 h (P < 0.05) with TCA; and 1.33, 2.13 and 2.58 h (P < 0.01) with tiopronin. Cmax showed the opposite behaviour with values (ng ml-1) normalized to the dose of 540 mg: 1235, 905 and 513 (P < 0.001) with TTPG; 888, 547 and 383 (P < 0.001) with TCA; and 7290, 6950 and 5170 (P < 0.01) with tiopronin.(ABSTRACT TRUNCATED AT 250 WORDS)

Antirheumatic drugs and macrophage function: effects on tumour cell growth in vitro.

The ability of the antirheumatic drugs D-penicillamine, chloroquine and levamisole to modify macrophage-mediated inhibition of tumour cell growth in vitro was investigated. Increasing numbers of rat peritoneal macrophages were cocultured with a fixed number of ascites hepatoma AH-13 rat tumour cells. Tumour cell growth was assessed as the uptake of 3H-thymidine (3H-TdR) by AH-13 cells at the end of a 24 h period of coculture with macrophages treated in vitro or in vivo with the various drugs. In vitro, preincubation of macrophages with D-penicillamine or chloroquine (50 - 250 micrograms/ml) increased tumour cell 3H-TdR incorporation, compared to cultures with untreated macrophages. Macrophages from rats treated with D-penicillamine or chloroquine (50 mg/kg/day orally) for 4 days similarly increased tumour cell 3H-TdR incorporation, compared to cultures with macrophages from untreated rats. These effects persisted for at least 3 to 4 weeks of treatment. Preincubation with levamisole (10 - 100 micrograms/ml) in vitro had no effect on macrophage-mediated inhibition of tumour cells, whereas increased tumour cell 3H-TdR incorporation was observed in cultures with macrophages from rats treated with levamisole (5 mg/kg/day orally) in vivo. Macrophages from rats with experimentally induced chronic inflammation, i.e. adjuvant arthritis, were found to increase tumour cell 3H-TdR incorporation, compared to macrophages from nonarthritic rats. This trend was further enhanced by treatment with D-penicillamine, chloroquine or levamisole.

Profile of activity of a new anti-inflammatory agent, ST 679 (MED 15).

ST 679 dose-dependently inhibited carrageenan-, concanavalin A-, and nystatin-induced oedema. Studies in rats with adjuvant arthritis showed that a long dosing regimen inhibited primary and secondary lesions. ST 679 was significantly active in reducing the severity of the already established disease and, when given in a short course at the time of adjuvant injection, permanently prevented the development of secondary lesions. Experimental allergic encephalomyelitis in guinea-pigs was not affected by ST 679.