Phospholipid‐Induced Changes of γ‐Aminobutyric Acid Transport in Cortex Grey Matter in Culture

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Abstract
The function of membrane phospholipids (PL) in the regulation of GABA transport and GABA carrier binding was investigated in organized cultures of rat cerebral cortex. The cellular lipid composition was changed by growing the cells in a delipidated nutrient solution or by short-term exposure of the cells to PL emulsions. Introduction of PL into the cellular matrix was monitored by analysis of biologically active fluorescently labeled phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Parinaroyl and dansyl derivatives were used. Conditions of maintenance as well as exogenously given PL affected the transport of GABA. Two transport systems were observed, 1 first-order system and 1 cooperative system. Saturated species of PC or PE reduced 1st-order GABA uptake with increase in chain length of the fatty acid residues. The effects of unsaturated PL were dependent upon the polar head. Unsaturated PC enhanced the capacity of the 1st-order transport of the amino acid. In comparison to cultures grown in lipid-free medium, introduction of diarachinoyl-PC into the cells increased the density of the 1st-order active transport sites by a factor of 8 and the affinity constant by a factor of 17. Diarachinoyl-PE reduced both kinetic parameters. GABA uptake via the cooperative system was enhanced by the unsaturated PE3 not by PC. The role of endogenous PL and their asymmetric distribution was studied by application of phospholipase A2, C and D. Stimulation of carrier activity was induced by hydrolysis of PL on the external leaflet. Inhibition occurred upon enzymatic degradation of external and cytoplasmic PL. Lipolysis also affected GABA receptor binding, suggesting that the effects observed represent the activity of both classes of binding sites, the carrier and the receptor. The latter accounted for a small fraction of the binding. Transport of the amino acid was temperature sensitive. The temperature curve was shifted within 2 discontinuities, appearing in the Arrhenius plot as a function of membrane lipids. Evidently, there is a partitioning of the proteins between fluid and ordered lipid domains. Displacement of the protein may govern the rate constants and/or the effective protein concentration.