PARTICIPACIÓN DE LAS PARTÍCULAS LIPÍDICAS EN LA LIBERACIÓN DE ENZIMAS EN CÉLULAS EN CULTIVO

Abstract

In pathological processes the highly selective permeability of the plasmatic membrane is affected, inducing among other things the release of enzymes into interstitial spaces and their subsequent incorporation into the bloodstream. However, the mechanism by which enzymes are released is unknown. Cone-shaped lipids present in lipid bilayers form transitory inducible molecular associations, known as lipoparticles, which are involved in fusion processes and the transport of ions and polar molecules across the lipid bilayer. Within the cell, enzymes are present in the cytoplasm and in the different organelles as well as associated to the cellular membrane and are released when the cell sustains damage of a nonlethal nature. Lipoparticle formation has been shown to be stimulated by inducers such as certain pharmaceutical agents and divalent cations. The goal of this study was to determine lipoparticle involvement in release of cytoplasmic enzymes in cell cultures of the human hepatocarcinoma G2 and cervical cancer C33 cell lines. Lipoparticle induction was carried out with chlorpromazine, procainamide, Ca2+ or Mn2+ at different concentrations for 45 min. Subsequently, cell viability and enzymatic activity in the supernatant were evaluated. Oxalic acid was used as a specific inhibitor of LDH to confirm activity of this enzyme and discard potential unspecific oxidation/reduction of the cell extract. Higher levels of released enzyme were obtained with chlorpromazine as this pharmaceutical is a potent lipoparticle inducer. Procainamide and Ca2+ and Mn2+ cations released lower levels of the enzyme in G2 cells. However, Mn2+ induced potent lipoparticle formation and higher levels of released enzyme (>50%) in C33 cells. In the presence of cyanide and iodoacetamide, which inhibit ATP synthesis in the respiratory chain and glycolysis respectively, lower levels of the enzyme (<20%) were released. However, cell damage was very evident, suggesting that cell viability was affected during treatment with these agents. Cyanide and iodoacetamide were incorporated with the cells and the latter were treated with different lipoparticle inducers, eliciting an increase in the levels of LDH, ALT and AST enzymes released into the medium in all cases and in both cell lines, which indicates that lipoparticle induction elicits enzyme release across membranes of the cell. Thus, enzyme release is not strictly dependent on hypoenergetic status as postulated so far. We therefore consider that inducing lipoparticle formation in the plasmatic membrane favors enzyme release without energy requirement. This may be a mechanism by which enzymes are released into the bloodstream in such a way that damage is not sustained by the cell membrane, as large amounts of lipoparticles may rupture the membrane, releasing its contents and eliciting cell death, a fact that may explain the high levels of serum enzymes in many pathologies. Understanding these mechanisms will enable the establishment of strategies for the improvement of pharmacological treatments aimed at prevention of lipoparticle formation and consequently enzyme release.