Aging and food restriction: effect on lipids of cerebral cortex.

In experimental animals dietary restriction reduces the body weight increase due to aging, increases longevity and delays the onset of age-related physiological deterioration, including age-related changes in serum lipids. Little is known about the influence of food restriction on brain lipids, whose concentration and composition have been shown to change with age. We studied whether some biochemical and biophysical parameters of rat brain membranes, known to be modified with age, were affected by a diet low in calories, in which 50% of lipids and 35% of carbohydrates have been replaced by fibers. The diet was started at weaning and maintained throughout the animal's entire life span. Animals fed the low calorie diet survived longer and gained less body weight than standard diet fed rats. Age-related increases in microviscosity, cholesterol/phospholipid and sphingomyelin/phosphatidylcholine ratios were reduced or restored to the levels of young animals in cortex membranes of 32 old rats fed the low calorie diet, while the age-related increase in mono- to polyunsaturated fatty acid ratios in phospholipids was further raised. In conclusion we have shown that a diet low in calories and high in fibers affects lipid composition in the rat brain, in a direction opposite to that normally believed to reduce age-related deterioration of brain functions.

Plasma and platelet lipid composition and platelet aggregation by arachidonic acid in women on the pill.

Sensitivity to induction of platelet aggregation by arachidonic acid (AA) and changes in plasma and platelet polyunsaturated fatty acid distribution were studied in seven women before and after six months of oral contraceptive (OC) treatment with a combination of d-norgestrel (0.25 mg) and ethinylestradiol (0.05 mg). Special interest was focused on AA because certain metabolites of fatty acid induce platelets to aggregate and are considered to play a crucial role in thromboembolic processes. In plasma, AA concentrations increased slightly, but significantly, in both the free fatty acid (FFA) and phospholipid fractions; in platelets AA increased in the phospholipid and neutral lipid fractions. The threshold aggregating concentration (TAC) of AA was significantly reduced in platelets of women after six months of OC treatment (0.65 +/- 0.08 versus 0.30 +/- 0.04 mM). This suggests that changes in platelet fatty acid composition may be associated with in vitro changes in platelet sensitivity to AA. Such changes may contribute to the thrombotic tendency associated with OC treatment.

PIP: Sensitivity to induction of platelet aggregation by (AA) arachidonic acid and changes in plasma and platelet polyunsaturated fatty acid distribution were studied in 7 women before and after 6 months of (OC) oral contraceptive treatment with a combination of d-norgestrel (0.25 mg) and ethinyl estradiol (0.05 mg). Special interest was focused on AA because certain metabolites of this fatty acid induce platelets to aggregate and are considered to play a crucial role in thromboembolic processes. In plasma, AA concentrations increased slightly, but significantly, in both the free fatty acid and phospholipid fractions; in platelets AA increased in the phospholipid and neutral lipid fractions. The threshold aggregating concentration of AA was signifcantly reduced in platelets of women after 6 months of OC treatment (0.65 + or - 0.08 versus 0.30 + or -0.04 mM). This suggests that changes in platelet fatty acid composition may be associated with in vitro changes in platelet sensitivity to AA. Such changes may contribute to the thrombotic tendency associated with OC treatment.

Modulation of phospholipid methylation in rat striatum by the corticostriatal pathway.

Surgical undercutting of cortical afferents and subsequent interruption of excitatory input increased phospholipid (PL) methylation (30-40%) in rat striatal synaptosomes as early as 3 days after lesion. The increase was still present 60 days after lesion, was protein-dependent and lesion-specific. The role of the dopaminergic agents on PL methylation in decorticated rats was investigated. In contrast with sham-lesioned rats, in which dopamine stimulated PL methylation, dopamine did not further stimulate phosphatidylethanolamine N-methyltransferase (PEMT) activity in decorticated rats. In addition, dopamine antagonists reversed the increase in PL methylation in lesioned rats at concentrations that had no effect in blocking DA-stimulated PL methylation in sham-lesioned rats. The specific antagonist of N-methyl-D-aspartate receptors, 2-amino-5-phosphonovaleric acid, increased PL methylation in non-lesioned striata, mimicking the effect of decortication, while N-methyl-D-aspartic acid counteracted the decortication-induced increase in PEMT activity. These data suggest a modulatory role of the cortex on PEMT activity in rat striatum.

Peripheral benzodiazepine receptor ligands in rat liver mitochondria: effect on cholesterol translocation.

Peripheral benzodiazepine receptors mediate cholesterol translocation between the outer and inner mitochondrial membranes in steroidogenic tissues. They are found in many other tissues too, including liver. We studied the effect of the peripheral benzodiazepine receptor ligands PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxa mid e], Ro 5-4864 (4-chlorodiazepam), hemin, protoporphyrin IX and N-methyl protoporphyrin IX on cholesterol mitochondrial intermembrane transport of cholesterol in vitro in rat liver. Endogenous cholesterol translocation from outer to inner mitochondrial membranes was significantly increased by PK11195 and N-methyl protoporphyrin IX (140% and 150% increase, respectively, at 1 microM, P<0.01). 5 microM protoporphyrin IX, 1 microM Ro 5-4864 and 5 microM hemin was ineffective. When mitochondria were labeled with exogenous [4-14C]cholesterol, PK11195 and N-methyl protoporphyrin IX were the most effective in increasing total cholesterol incorporation and cholesterol translocation into inner membranes, and their effect was dose-dependent. These data suggest that in liver the binding to peripheral benzodiazepine receptors is related to cholesterol translocation and the interaction of ligands with these receptors may play a role in the complex mechanism of regulation of cholesterol traffic between liver mitochondrial membranes.

Peripheral benzodiazepine receptor ligands in rat liver mitochondria: effect on 27-hydroxylation of cholesterol.

The effect of peripheral benzodiazepine receptor ligands: PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)isoquinoline-3-carboxamid e), Ro 5-4864 (4-chlorodiazepam), hemin, N-methyl protoporphyrin IX and protoporphyrin IX on liver mitochondrial 27-hydroxylation of cholesterol was studied by adding them together with [4-14C]cholesterol. N-Methyl protoporphyrin IX, PK11195 and protoporphyrin IX stimulated mitochondrial 27-hydroxylation of [4-14C] cholesterol in vitro, the first two being the most potent (2-3-fold increase). Ro 5-4864 and hemin were not active. 27-Hydroxylation of [4-14C]cholesterol was reduced to below control levels (respectively 40 and 56% decrease compared to control, P < 0.01) when PK11195, N-methyl protoporphyrin IX or protoporphyrin IX were allowed to equilibrate in vitro with mitochondria for 20 min at 37 degrees C. Hepatic protoporphyria was induced using 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) (100 mg/kg, i.p.) to study the effect of in vivo accumulation of large amounts of dicarboxylic porphyrins, i.e. endogenous peripheral benzodiazepine receptor ligands, on cholesterol 27-hydroxylation. DDC treatment caused an increase in total porphyrin content in liver homogenate (10-fold) and mitochondria (2-fold). Mitochondrial 27-hydroxylation of [4-14C]cholesterol was depressed after treatment (60% decrease, P < 0.01). We suggest that peripheral benzodiazepine receptor ligands act on liver mitochondrial 27-hydroxylation of cholesterol by a mechanism coupled to these receptors and that the time of exposure of peripheral benzodiazepine receptors to ligands is a major factor. The modulation of 27-hydroxycholesterol production may have a physiological role in liver and possibly in other tissues.

Effect of piribedil and its metabolite, S584, on brain lipid peroxidation in vitro and in vivo.

We studied the effect of piribedil (1-3,4-methylendioxybenzyl-4-(2-pyrimidyl) piperazine) and its catechol metabolite, S584 (1-(3,4-dihydroxybenzyl-4-(2-pyrimidinyl)-piperazine), on rat brain lipid peroxidation (a) in vitro in rat synaptosomes and cortical slices after induction of an oxidative stress and (b) in vivo in mouse brain after short-term exposure (two and three 4-h cycles) to O2/CO2 (95%:5%). The metabolite (10[-4]-10[-5] M), but not piribedil, prevented Fe3+-stimulated lipid peroxidation in rat synaptosomes and in rat cortical slices incubated with high oxygen concentrations. Piribedil (7.5 and 30 mg/kg, orally), counteracted the increase in thiobarbituric reactive substances in the brain of mice only when these were exposed to two or three cycles of a high oxygen concentration. S584 (30 mg/kg, orally) reduced thiobarbituric acid reactive substances in brain in mice exposed either to air (control) or to three cycles of a high oxygen concentration. These results suggest that piribedil has an antiperoxidative effect in brain, which may be partly related to the in vivo formation of the catechol metabolite, S584.

Comparison of hexachlorobenzene-induced alterations of microsomal membrane composition and monooxygenase activity in male and female rats.

The effect of hexachlorobenzene (HCB) on microsomal cytochromes P-450 and b5, monooxygenase activity and membrane composition was examined in male and female Fischer rats. Cytochrome P-450 was induced more in male than in female animals while cytochrome b5 was induced only in males. Analysis of patterns of induction of microsomal monooxygenases showed that aminopyrine-N-demethylase activity doubled in both sexes after treatment while aryl hydrocarbon hydroxylase activity was 16 times the control value in the females and 1.5 times in the males. After HCB treatment the phospholipid content of microsomal membranes per gram of liver was increased in both sexes while cholesterol was unchanged. Analysis of the phospholipids (PL) pattern showed that the percentage of sphingomyelin (SPH) decreased significantly (50% of the control value) while phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) did not change. These changes resulted in a reduction of membrane microviscosity and indicate that HCB interferes with the biosynthesis of phospholipids containing choline. Free fatty acid (FFA) content also dropped in both sexes but females were more affected; free arachidonic acid rose in females. HCB induction of microsomal cytochromes and monooxygenases is thus accompanied by marked modifications of membrane composition. Comparing the 2 sexes, HCB showed more pronounced features of 'PB type' inducers in males.

Phospholipid composition, phosphoinositide metabolism and metastatic capacity in murine melanoma B16 variants at different stages of growth.

Experimental efforts to identify characteristic features of metastatic subpopulations have led to the selection of strains of specialized cells with high and low metastatic potential in the hope that by studying their biochemical and biophysical properties we might start to clarify how tumour cells metastasize. We report data on the phospholipid composition of three variants of murine melanoma B16: F1, with low metastatic potential; F10, highly metastatic when injected i.v.; and BL6, highly metastatic, spontaneous metastases developing from a primary s.c. tumour. Cells were studied at different stages of growth: subconfluent cultures (40-70 x 10(3) cells/cm2) or dense cultures (140-170 x 10(3) cells/cm2). Total phospholipid content decreased as cell density increased in all variants; these changes can probably be related to the reduction in cell volume with increasing cell numbers in the well. As a consequence of this reduction, the amounts of individual phospholipids also decreased in dense cultures. Phosphatidylinositol behaved differently in the highly metastatic variants. In the F1 strain it was three times lower than would be expected from the total phospholipid reduction, while in F10 and BL6 levels increased when cell density increased. Differences in phosphatidylinositol level were also found between variants within each density, suggesting that phosphoinositide synthesis may be related to the metastatic potential of the variants. Incorporation of ([3H] myo)-inositol incorporation into phospholipids over a period of 4 h was greater in F1 cells than in F10 and BL6 at both cell densities.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects of benzodiazepines on phospholipid methylation in hippocampus and cerebellum of rats.

To elucidate the relationship between the occupancy of BDZ binding sites and phospholipid methylation in brain, we examined phosphatidylethanolamine-N-methyltransferase (PEMT) activity in synaptosomes of rat hippocampi and cerebella in the presence of BDZ ligands with different modes of action. We found that Ro 5-4864, a specific ligand for "peripheral type" receptors, increased PL methylation in hippocampal and cerebellar synaptosomes. This effect was directly related to receptor occupancy, since the specific antagonist PK 11195 inhibited the rise in PEMT activity induced by Ro 5-4864. Clonazepam, on the other hand, tended to reduce PL production in cerebellum and hippocampus except for hippocampal (3H)-phosphatidyl-N-monomethylethanolamine which was elevated by 40 to 70% at doses ranging from 10(-9) to 10(-6) M. When equimolar concentrations of the antagonist Ro 15-1788 were given in association the clonazepam-induced phosphatidyl-N-monomethylethanolamine increase was reduced by 70%. These data support the involvement of structural and functional membrane alterations in the action of BDZ.

Adriamycin causes hyperlipemia as a consequence of nephrotoxicity.

Adriamycin induced hyperlipemia: its features and mechanism(s) in rats were investigated. Massive hyperlipemia occurred 14-21 days after a single dose of adriamycin (7.5 mg/kg i.v.). All lipoprotein fractions were affected. Mild but significant changes in tissues were observed (liver and intestine triglycerides and kidney phospholipids were reduced). Lipid synthesis and secretion was decreased, as shown by the Triton WR1339 test 7 days after treatment, but subsequently returned to normal. Mitochondrial oxidation of long-chain fatty acids was markedly reduced in kidney, and a slight reduction was also observed in heart. Lipoprotein lipase activity was reduced in adipose tissue. These results suggest that adriamycin hyperlipemia is due to reduced lipid storage and utilization. Carnitine did not counteract hyperlipemia and proteinuria after adriamycin. Analogies to hyperlipemia following puromycin aminonucleoside-induced nephrotoxicity are discussed.

Accumulation and metabolism of uneven fatty acids present in single cell protein.

Liquipron and Toprina, obtained by growing yeasts (Candida maltosa and Candida lipolytica) on n-hydrocarbons, were investigated to ascertain the biological significance and possible toxicological implications of their high content of uneven fatty acids (UFA). It was confirmed that the extent to which UFA accumulate in adipose tissue of rats fed the 2 products reflects only partially their UFA contents. The presence of UFA in rat tissues does not appear to alter intermediate metabolism. The capacity of liver mitochondria ot oxidize palmitic acid was similar in control and in Liquipron-treated rats. Palmitic acid and heptadecanoic acid did not compete for oxidation when mixed at concentrations which reflect their presence in the tissues of animals fed high levels of Liquipron.

Piracetam: physiological disposition and mechanism of action.

Piracetam, the prototype of nootropic drugs, is claimed to have therapeutic value in some patients suffering from cognitive deficits, especially when hypoxia may be a factor in the deficits. The drug lacks sedative or stimulatory effects and apparently does not influence behavior in normal people. The site of action and the mechanism by which piracetam exerts its beneficial effects are not well established. This review has summarized experimental and clinical data on its absorption, distribution, and excretion, and described the biochemical data available that bear on its mechanism of action.

A novel pharmacological approach for paraquat poisoning in rat and A549 cell line using ambroxol, a lung surfactant synthesis inducer.

Paraquat (PQ) is a widely used herbicide that causes acute adult respiratory distress syndrome (ARDS) and chronic lung damage (diffuse fibrosis). One of the earliest biochemical effects induced by PQ is damage to type II pneumocytes with consequent depletion of surfactant. With the aim of counteracting the toxic effects of PQ, a series of investigations were performed into the possible protective effect of the drug ambroxol, which induces the synthesis of surfactant in lung alveolar type II cells. The number of survivors and survival time of rats treated ip with 35 mg PQ/kg was significantly increased by 3 days of ambroxol pretreatment and by ambroxol treatment 30 min or 2 hr after PQ. Total phospholipid content in lung and bronchoalveolar lavage fluid (BALF) was significantly reduced 30 hr after treatment with PQ alone. The association of ambroxol with PQ significantly antagonized this reduction. In BALF the ratio between palmitic acid and stearic acid concentrations was significantly lower in animals treated with PQ alone but was returned to normal by the association with ambroxol. The cell line A549, exposed in vitro to PQ concentrations from 0.5 x 10(-4) to 2 x 10(-3) M, showed a significant dose-dependent loss of viability. Cells pretreated with ambroxol (10 mg/ml) were more resistant to PQ and their viability started to decrease significantly only from a PQ concentration of 0.8 x 10(-3) M. Membrane microviscosity was measured on the same cells. Cells treated with PQ alone showed a reduction of membrane microviscosity, which was significantly counteracted by ambroxol pretreatment. The curves of modification of membrane microviscosity of cells treated with PQ and with ambroxol plus PQ paralleled those of cell viability, indicating that the stimulation of surfactant synthesis in vitro may be a prerequisite for counteracting some of the early effects of PQ.

Effect of cell density on cytotoxicity of ether lipid analogues in variants of B16 murine melanoma.

Ether lipids are defined here as analogues of naturally occurring lysophosphatidylcholines with cytotoxic activity against neoplastic cells. The activity of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET18OMe) and 3-hexadecylmercapto-2-methoxymethyl-propyl-1-phosphocholine (Ilmofosine) (BM 41.440) was tested in variants of B16 murine melanoma, grown in adhesion cultures (B16F1 with low metastatic potential; B16F10 and B16BL6 with high metastatic potential). Cytotoxicity was evaluated by counting the cells that survived after 24 h of drug exposure. Cholesterol, sphingomyelin, total phospholipid and phosphatidylcholine levels were determined. After 24 h of drug exposure, cultures of the B16BL6 variant contained a larger number of cells, especially when high drug concentrations (100-250 microM) were used, than cultures of the B16F1 and B16F10 variants. The sensitivity to ET18OMe of the three variants was evaluated at different cell densities (at each density the dose was equalized per number of cells/well; 0.1 mumol/10(6) cells/well). In B16F1 and B16F10 cultures the dose-response curve was not affected by the number of cells/well, while in B16BL6 no more than 20% of the cells were killed at all cell densities measured. A linear relationship was noted between cell density and cholesterol/phospholipid and sphingomyelin/phosphatidylcholine ratios in the resistant variant B16BL6, confirming that lipid composition modulates the cytotoxic activity of ether lipids.

Physiological disposition of oral piracetam in Sprague-Dawley rats.

The distribution and fate of piracetam (2-oxo-1-pyrrolidine acetamide, Nootropil), the prototype 'nootropic drug', was examined in rats given 100-1000 mg kg-1 by gavage, with or without [3H]piracetam as a tracer. Peak serum concentrations were attained after 60 min. Its half-life of disappearance from serum was about 2 h during the initial 8 h after administration and then about 6.4 h for the next 16 h. Brain piracetam concentrations equilibrated with those of serum at about 4 h, after which they fell exponentially but remained about twice those of serum; piracetam concentrations in the brainstem were lower (by 30-40%) than those in the cortex, olfactory bulb, and colliculi. No evidence could be obtained for significant piracetam metabolism, either in-vivo or when incubated with liver homogenates. No specific binding of [3H]piracetam to any of various subcellular fractions was observed after its administration along with unlabelled carrier. Repeated daily doses of piracetam (7 days, 100 mg kg-1) failed to elevate serum or brain concentrations beyond those observed after a single dose.

Intraluminal inflammation in the airways of patients with chronic bronchitis after treatment with Ambroxol.

Ambroxol is a mucus-modifying drug with a known ability to stimulate surfactant secretion and inhibit, in vitro, the production of proinflammatory cytokines, neutrophil chemotaxis, and Na+ absorption by the airway epithelium. In dogs inhaling ozone, bronchial hyperreactivity can be inhibited by aerosolized Ambroxol. To verify the possibility of producing anti-inflammatory effects in a clinically relevant condition, 20 patients with chronic bronchitis, randomly divided into two balanced matched groups, were submitted to a 14 day trial with Ambroxol, 150 mg.day-1, or placebo, according to a double-blind design. A bronchoalveolar lavage (BAL) was performed the day before starting treatment (T0) and at the 14th day (T14). The analysis of the cellular and soluble (total proteins, albumin, immunoglobulin G and A (IgG and IgA)) BAL components demonstrated no clear modifications. In particular, neutrophil values from the bronchial aliquot showed a large dispersion, with no significant differences (Ambroxol: T0 = 13.7 +/- 5.2%, T14 = 14.0 +/- 6.8%; placebo: T0 = 3.6 +/- 1.1%, T14 = 5.5 +/- 2.2%). We found a nonsignificant increase of phospholipids in BAL supernatants from Ambroxol-treated patients (2.5 +/- 1.9 vs 3.0 +/- 1.9 micrograms.mg-1 of protein); whilst in the placebo group data before and after treatment were superimposable (2.2 +/- 1.5 vs 2.3 +/- 1.9 micrograms.mg-1 of protein). In conclusion, we have failed to demonstrate that conventional treatment with oral Ambroxol can modify surfactant and BAL cell populations in the airways of patients with chronic bronchitis.