José Manuel Martínez-Martos⁽¹⁾, María Jesús Ramírez-Expósito⁽²⁾, María Dolores Mayas-Torres⁽³⁾, María Jesús García-López⁽⁴⁾, Isabel Prieto-Gómez⁽⁵⁾, Garbiñe Arechaga-Maza⁽⁶⁾, Manuel Ramírez-Sánchez^{(7)
(1)(3)(4)(5)(6)(7)}Unit of Physiology. University of Jaén - Jaén. Spain
⁽²⁾Unit of Physiology. University of Jaen - Jaén. Spain

	[Cell Biology & Cytology]	[Endocrinology]	[Neuroscience]	[Physiology]

INTRODUCTION

In recent years, evidence has been accumulating that in addition to the well-established second messengers, the existence of additional messenger molecules should be taken into account ⁽¹⁾. Several studies carried out in various cell types suggest that fatty acids act as second messengers and modulators by satisfying the criteria through their capacity to modulate some enzymes as phospholipases, protein kinases, G-proteins, adenylate and guanylate cyclases activities as well as ion channels and other biochemical events involved in stimulus-response couple mechanisms ^(2-5).

In this way, fatty acids can act as second messengers involved in the transduction of external signals because their concentrations are rapidly and transiently altered in response to the binding of a specific agonist to plasma membrane receptors and the substitute for the classical second messengers of the inositide phospholipid and the cyclic AMP signal transduction pathway.

Although a number of studies have addressed the interaction between fatty acids and lipids with CNS peptides, a comprehensive overview has not yet been provided. A major problem confronting a compelling delineation of these issues derives from the fact that fatty acids and lipids exist in large quantities in the body. They are present in every cell in the body and they interact with various components of the cell, mainly the membrane. In contrast, brain peptides are produced in small quantities and they affect and interact with a limited number of cell types.

In general, fatty acids and peptides may interact in one of more of the following ways: synthesis, release, lipid metabolism, enzymes, receptors and post receptor events ⁽⁶⁾.

In this way, aminopeptidases (AP) are generally zinc-metalloenzymes which hydrolyse peptide bonds near the N-terminal end of peptides and polypeptides. The importance of these enzymes is based on their major role in the metabolism of proteins and in the regulation of circulating hormones and biologically active peptides in tissues ⁽⁷⁾. The aim of the present work is to study the behaviour of several AP expressed in whole cell primary astrocyte cultures of the frontal cortex of the rat, after incubation with several concentrations of oleic and linoleic fatty acids and cholesterol in the culture medium. It has been demonstrated that primary cultures of nervous cells are a useful model for analysing cellular functions of defined populations, and therefore represent a valuable tool for studying functional and biochemical aspects of cell physiology ⁽⁸⁾.

MATERIAL & METHODS

Primary cultures of astrocytes from 21-day-old fetuses were prepared from brain hemispheres as described ^(9,10). The experimental procedures for animal use and care were in accordance with the European Community Council Directive 86/609/EEC. Fetuses were obtained under sterile conditions from rats and frontal cortex were dissected free of meninges and mechanically dissociated in Dulbecco´s modified Eagle´s medium (DMEM). The cell suspension was vortexed at maximum speed, filtered through a nylon mesh of 80 µm pore size and resuspended in DMEM containing 20% fetal calf serum (FCS) and 1% antibiotics. Every three days in culture, half the medium was replaced until the cultures were confluent (approximately 14 days). After this time, cells were detached and the cell suspension was divided in different tubes; several concentrations of oleic and linoleic fatty acids and cholesterol (1µM, 10 µM and 100 µM) were added. Water-soluble complexes of fatty acids and cholesterol with methyl-ß-cyclodextrin as carrier were used to facilitate the dissolution into the culture medium. The control groups were incubated only with the correspondent amounts of methyl- -cyclodextrin. 50 µL of these cell suspensions were cultured in 96-well plates (Costar) (1.5x105 cells/well) and incubated in a humidified atmosphere of 5% CO2 at 37ºC. The astrocyte cultures contained more than 95% of positive cells for the glial fibrillary acidic protein (GFAP). After 3-4 days of incubation (>90% of confluence) AlaAP, ArgAP, CysAP, LeuAP, TyrAP, and pGluAP activities were analysed in whole cells, using as substrates Alanyl- -naphthylamide (AlaNNap), arginyl- -naphthylamide (ArgNNap), cistinyl- -naphthylamide (CysNNap), Leucyl- -naphthylamide (LeuNNap), tirosyl- -naphthylamide (TyrNNap), and piroglutamyl- -naphthylamide (pGluNNap) in accordance with the modified methods of Greenberg ⁽¹¹⁾, Schwabe and McDonald ⁽¹²⁾ and Wagner et al. ⁽¹³⁾. After eliminate the culture medium, the cells were incubated during 30 min at 37ºC with 100 µL of the incubation solution which contain 100 µM of AlaNNap, ArgNNap, CysNNap, LeuNNap, TyrNNap or pGluNNap in artificial cerebrospinal fluid (CSF) (NaCl 116mM, KCl 5.4 mM, MgCl2 0.9 mM, CaCl2 1.8 mM, NaHCO3 25 mM, glucose 10mM) pH 7.2. All the reactions were stopped by addition of 100µl of acetate buffer 0.1M (pH 4.2) containing fast garnet GBC salt and Tween-20. The amount of -naphthylamine released as the result of the enzymatic activity was coupled with the GBC salt appearing a red complex which can be spectrophotometrically determined at 550 nm ⁽¹⁴⁾. The proteins were quantified in triplicate by the method of Bradford ⁽¹⁵⁾, using BSA as a standard. The specific aminopeptidase activities were expressed as nmol of AlaNNap, ArgNNap, CysNNap, LeuNNap, TyrNNap and pGluNNap hydrolysed per minute per mg of protein, using a standard curve prepared with the latter compound under corresponding assay conditions. The spectrophotometric assay was linear with respect to time of hydrolysis and protein content.

The morphological features of astrocytes in primary culture were checked using phase-contrast microscopy and Giemsa staining.

Statistical analysis.

We used one-way analysis of variance (ANOVA) to analyse differences between groups. Post-hoc comparisons were made using Newman-Keuls test. P-values below 0.05 were considered significant.

RESULTS

Values (mean±SEM) of specific aminopeptidase activities in cultured astrocytes added with several concentrations of oleic and linoleic fatty acids and cholesterol are presented in Figure 1. Oleic and linoleic fatty acids decrease AlaAP activity in a concentration-dependent manner, whereas cholesterol does not modify AlaAP activity. Thus, oleic acid 1 µM decreases significantly (P<0.01) AlaAP activity in 12% under control, whereas oleic acid 10 µM and 100 µM inhibits AlaAP activity in 19% under control (P<0.01). Linoleic acid decreases AlaAP activity in 8% under control (P<0.05); 10 µM linoleic acid decreases AlaAP activity in 16% under control (P<0.01) and 100 µM linoleic acid inhibits AlaAP activity in 25% under control (P<0.01).

With regard to the ArgAP activity, oleic acid does not modify this activity, whereas linoleic acid decreases ArgAP activity in 43% (P<0.01) only with the higher concentration of fatty acid tested. On the contrary, cholesterol increases ArgAP activity in a concentration-dependent manner. Thus, cholesterol 10 µM increases ArgAP activity in 23% over control (P<0.01), and cholesterol 100 µM in 67% over control (P<0.01), although cholesterol 1 µM does not modify ArgAP activity.

Oleic acid modifies CysAP activity when low quantities are added to the culture medium. In this way, oleic acid 1 µM decreases CysAP activity in 23% under control (P<0.01), whereas oleic acid 10 µM decreases CysAP activity in 16% under control (P<0.01). On the contrary, oleic acid 100 µM does not modify CysAP activity. By other hand, linoleic acid does not modify CysAP activity at any of the concentrations tested, but cholesterol increases significantly CysAP activity. in a concentration-dependent manner. Thus, cholesterol 1 µM increases CysAP activity in 6% over control (P<0.01), cholesterol 10 µM increases in 16% over control (P<0.01) and cholesterol 100 µM increases in 34% over control.

LeuAP activity is modified by oleic acid, decreasing in 30% under control with oleic acid 1 µM, in 38% under control with oleic acid 10 µM and in 32% under control with oleic acid 100 µM (P<0.01 in all cases). On the contrary, linoleic acid does not modify LeuAP activity. Moreover, cholesterol does not modify LeuAP activity in cultured astrocytes except when a high concentration is added (cholesterol 100 µM), which increases slightly LeuAP activity (5%; P<0.01).

With regard to the TyrAP activity, oleic acid does not modify this activity, but linoleic acid decreases significantly TyrAP activity in 30% under control (P<0.01), independently of the concentration of linoleic acid added to the culture medium. On the contrary, cholesterol increases TyrAP activity in a concentration-inversely dependent manner. Thus, cholesterol 1 µM increases TyrAP activity in 64% over control, cholesterol 10 µM increases TyrAP activity in 53% over control and cholesterol 100 µM increases TyrAP activity only in 43% over control (P<0.01 in all cases).

Neither oleic acid nor linoleic acid modifies pGluAP activity at any of the concentration tested. On the contrary, cholesterol increases pGluAP activity in a concentration-dependent manner. Thus, cholesterol 1 µM increases pGluAP activity in 16% over control, cholesterol 10 µM increases in 25% over control, and cholesterol 100 µM increases pGluAP activity in 57% over control (P<0.01 in all cases).

DISCUSSION

The present study clearly shows that the addition of oleic and linoleic fatty acids to the culture medium modifies several aminopeptidase activities in cultured astrocytes, decreasing their activity. On the contrary, the presence of exogenous cholesterol in the medium increases aminopeptidase activities.

To our knowledge, little is known about the effects of fatty acids and/or cholesterol added into the culture medium on aminopeptidase activities. Reliable results from a number of research laboratories have established that the modifications in the level of fatty acids is able to change the entire profile of fatty acids as well as the cholesterol level in the cellular membranes ^(16-18). In this way, Murphy ⁽¹⁹⁾ has demonstrated that cultured astrocytes take up exogenous linoleic acid and incorporate its metabolites into phospholipids, and that the resulting changes in membrane fatty acids composition modify only specific cell functional properties. These chemical changes can be accompanied by changes in the physical state of the membrane, and could be responsible of the effects of fatty acids on AP activities reported here. Moreover, it is well known that changes in the relative amounts of free fatty acids in the cellular membrane may be a major factor in the physiological role of the membrane by changing the membrane fluidity ⁽²⁰⁾. In synaptosomal membranes from rat and monkey brain cortex, the addition of oleic acid decrease the microviscosity of the membrane core in 7-10% and of the membrane surface in 5-7%, being able to modify the opioid receptor binding ⁽²¹⁾. Furthermore, in vivo experiments administrating a high-fat dietary supplementation with olive oil, rich in oleic acid, showed an influence on several aminopeptidases in serum and different tissues of mice ^(22,23). Thus, the olive oil-fed group had significantly higher ArgAP activity levels in serum than control. Soluble AlaAP and ArgAP activities increased significantly in the brain, adrenal gland and testis of olive oil-fed animals. Soluble CysAP activity increased significantly in testes and liver and decreased in the adrenal glands of olive oil-fed mice. pGluAP activity decreased in the adrenal gland of high fat-fed animals. These findings support that a diet supplemented with olive oil modifies certain aminopeptidase activities in specific tissues, indicating that aminopeptidases could be modulated by fatty acids through mechanisms involving membrane fluidity and specific interactions with membrane constituents.

By other hand, cholesterol is a major molecule in the membrane and an elevated cholesterol level results in a decrease in the membrane fluidity and in disturbances of the membrane function also. In addition, steroids are derivatives of cholesterol. Previous in vitro results reported by us demonstrated a direct influence of cholesterol and steroid hormones on aminopeptidase activities ⁽²⁴⁾. Astroglial cells possess both receptors for sex steroid hormones and enzymes for their metabolism ^(25,26). On this way, fatty acids such as oleic acid modulate steroid hormone receptors in the brain, inhibiting the binding between the steroid and its cytosolic receptor in the rat cortex ⁽²⁷⁾. Moreover, recent evidence indicates that astroglia may be involved in the synthesis of endogenous neurosteroids, which regulate the morphology and/or GFAP distribution of astrocytes ⁽²⁸⁾.

By other hand, many studies have demonstrated the role of specific fatty acids, cholesterol and other steroids on all aspects of synthesis, release and receptor functions of several peptide systems ⁽²⁹⁾. In this way, several examples have been reported: substance P decreases the levels of linoleic acid, increases the cholesterol level, and decreases the level of membrane fluidity ^(30,32). Thyrotrophin-releasing hormone (TRH) inhibits lipid peroxidation ⁽³³⁾ and increases the level of arachidonic acid in blood and brain ^(34,35). Arachidonic acid also increases membrane fluidity ^(36,37). Oleic acid inhibits the release of GHIF ⁽³⁸⁾. Polyunsaturated fatty acids improve the binding of -endorphin to its receptor ^(39-41). Similar findings were found for enkephalin binding ⁽⁴²⁾. A complex pattern of peptide-peptide and peptide-fatty acid interactions may be therefore, observed. Certain fatty acids are particularly active elements in the peptides-fatty acid interaction. For example, linoleic acid increases the release of LH and GH ^(43-46) while substance P decreases the level of this fatty acid ⁽³⁰⁾. By other hand, our results showed an important increase on ArgAP, CysAP, TyrAP and pGluAP activities when cholesterol is added to the culture medium. Cholesterol is involved in many functions of the membrane. As previously cited, it is well established that cholesterol decreases the membrane fluidity index, with consequences on the activity of membrane-bound enzymes, ion channels and receptors functions. In addition, cholesterol is involved in dopamine release. Moreover, cholesterol is a key molecule in the end product of the CRF-ACTH axis. Steroids are derivatives of cholesterol and it is therefore interesting that various fatty acids have differential effects on cholesterol metabolism.

Huang et al. ⁽⁴⁷⁾ cite many studies which show that n-6 series of fatty acids are able to reduce the level of cholesterol in the blood serum. The data of Horrocks and Harder ⁽⁴⁸⁾ showed that n-6 fatty acids and n-3 fatty acids differ in their mode of action in cholesterol reduction; n-6 fatty acids redistribute cholesterol while n-3 fatty acid actually reduces the level of cholesterol.

Special attention must be appointed on the differential effects of oleic and linoleic fatty acids on AP activities. Thus, whereas oleic acid inhibits CysAP and LeuAP, linoleic acid inhibits ArgAP and TyrAP. Only AlaAP is inhibited by both oleic and linoleic fatty acids. Various fatty acids have a different role in the nervous system and in the body. Salem claimed that the nervous system has an absolute molecular species requirement for proper function ⁽⁴⁹⁾. Our studies confirm this statement; moreover, it could be possible that a proper ratio between fatty acids must be required. It has been described that the ratio of n-3 and n-6 may be a key factor in modulating behavioural and neuropharmacological effects of fatty acids ⁽⁵⁰⁾.

Therefore, it seems that various fatty acids and lipids play a major role in the synthesys, release, and function of several peptides, and are not -nonspecific- molecules which act as regulators of the peptide system.

Until now, has been reported that fatty acids and peptides may interact at the level of synthesis, release, receptors and post-receptors events. On the contrary, although it has been demonstrated that fatty acids and lipids can modify the level of activity of certain membrane-bound enzymes, it is unresolved whether those enzymes are important to the peptides activity. The changes reported here on aminopeptidase activities due to cholesterol and fatty acids clearly demonstrate that these substances can modulate peptides activity through their degradation route due to aminopeptidases.

Aminopeptidase activities play a major role in the regulation of several biologically active peptides. AlaAP may hydrolyse bradykinins ⁽⁷⁾ and enkephalins ⁽⁵¹⁾ and may also act as an angiotensinase ⁽⁵²⁾. ArgAP activity specifically hydrolyses basic N-terminal residues from peptides and arylamide derivatives ⁽⁵³⁾. Because of its exopeptidase activity, it has been implicated in the metabolism of met-enkephalin ⁽⁵⁴⁾ and angiotensin III ⁽⁵²⁾; its endopeptidase activity is also thought to be involved in neurotensin metabolism ⁽⁵⁵⁾. CysAP activity has been reported to hydrolyse oxytocin and vasopressin ⁽⁵⁶⁾. Although several peptides have been proposed as susceptible substrates for pGluAP in vitro, the most important endogenous substrate may be the hypophysiotropic hormones TRH ⁽⁵⁷⁾ and GnRH ⁽⁵⁸⁾, although neurotensin and bombesin have been reported also. LeuAP may hydrolyse enkephalins ⁽¹⁴⁾, dinorphins ⁽⁵⁹⁾ and substance P ⁽⁶⁰⁾. TyrAP has been described as an enkephalin aminopeptidase ⁽¹³⁾. The purified enzyme hydrolyses the aminoterminal tyrosine residue of met-enkephalin ^(13,14). It is important to note that AP have been usually described as non-specific enzymes that are capable to hydrolyse a broad extent of endogenous peptide substrates and arylamide derivatives in different degree. However, our results, using whole cells without homogenization, demonstrate different patterns of AP activity in response to the addition of oleic and linoleic fatty acids and cholesterol into the culture medium. It could be possible that in vivo, AP activities would be extremely specifics on their native substrates and modulate them strictly depending on the microenvironment surrounding the cell type.

Therefore, the present results demonstrate that oleic and linoleic fatty acids and cholesterol influence aminopeptidase activities in primary astrocytes when added to the culture medium. These effects may induce functional modifications in the action of aminopeptidases on susceptible substrates; alternatively, they may reflect concomitant modifications in susceptible endogenous substrates.

Finally, it has been seem recently that oleic acid causes a dose-dependent inhibition of GAP junctions permeability in cultured rat astrocytes. The authors suggest that oleic acid may be a physiological mediator of the transduction pathway leading to the inhibition of intercellular communication ⁽⁶¹⁾. Due to the paracrine and/or autocrine functions of most of biologically active peptides, the inhibition of several AP activities by fatty acids may be a reflect of those mechanism that modulates intercellular communication, and must be also considered.

ACKNOWLEDGEMENTS

We wish to thank Dr. W. Rzeski (Experimental Anaesthesiology, University Hospital Charité, Humboldt University, Berlin, Germany) for initiating us into the primary culture of neural cells.

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Discussion Board

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[ABSTRACT] [INTRODUCTION] [MATERIAL & METHODS] [RESULTS] [FIGURES] [DISCUSSION] [ACKNOWLEDGEMENTS] [BIBLIOGRAPHY] [Discussion Board]

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	[Cell Biology & Cytology]	[Endocrinology]	[Neuroscience]	[Physiology]

José Manuel Martínez-Martos, María Jesús Ramírez-Expósito, María Dolores Mayas-Torres, María Jesús García-López, Isabel Prieto-Gómez, Garbiñe Arechaga-Maza, Manuel Ramírez-Sánchez
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Last update: 16/12/99