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]

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.

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