Glycoalkaloids selectively permeabilize cholesterol containing biomembranes.

The effects of the glycoalkaloids alpha-solanine, alpha-chaconine and alpha-tomatine on different cell types were studied in order to investigate the membrane action of these compounds. Hemolysis of erythrocytes was compared to 6-carboxyfluorescein leakage from both ghosts and erythrocyte lipid vesicles, whereas leakage of enzymes from mitochondria and the apical and baso-lateral side of Caco-2 cells was determined. Furthermore, the effects of glycoalkaloids on the gap-junctional communication between Caco-2 cells was studied. From these experiments, it was found that glycoalkaloids specifically induced membrane disruptive effects of cholesterol containing membranes as was previously reported in model membrane studies. In addition, alpha-chaconine was found to selectively decrease gap-junctional intercellular communication. Furthermore, the glycoalkaloids were more potent in permeabilizing the outer membrane of mitochondria compared to digitonin at the low concentrations used.

Molecular basis of glycoalkaloid induced membrane disruption.

In this study the interaction between the glycoalkaloids alpha-chaconine, alpha-solanine and alpha-tomatine and sterols in model membranes was analysed systematically using techniques like membrane leakage, binding experiments, detergent extraction, electron microscopy, NMR and molecular modelling. The most important properties for sterols to interact with glycoalkaloids turned out to be a planer ring structure and a 3 beta-OH group, whereas for alpha-chaconine the 5-6 double bond and the 10-methyl group were also of importance. The importance of sugar-sugar interactions was illustrated by the high synergistic effect between alpha-chaconine and alpha-solanine, the leakage enhancing effect of glycolipids, and the almost complete loss of activity after deleting one or more mono-saccharides from the glycoalkaloids. The formed complexes which were resistant against detergent extraction existed of glycoalkaloid/sterol in a 1:1 ratio and formed tubular structures (alpha-chaconine) with an inner monolayer of phospholipids, whereas with alpha-tomatine also spherical structures were formed. Based on the results a molecular model for glycoalkaloid induced membrane disruption is presented.

Dual specificity of sterol-mediated glycoalkaloid induced membrane disruption.

In this study the effects of the glycoalkaloids alpha-solanine, alpha-chaconine, alpha-tomatine and the aglycone solanidine on model membranes composed of PC in the absence and presence of sterols have been analysed via permeability measurements and different biophysical methods. The main result is that glycoalkaloids are able to interact strongly with sterol containing membranes thereby causing membrane disruption in a way which is specific for the type of glycoalkaloid and sterol. For this dual specificity both the sugar moiety of the glycoalkaloid and the side-chain of the sterol on position 24 turned out to be of major importance for the membrane disrupting activity. The order of potency of the glycoalkaloids was alpha-tomatine > alpha-chaconine > alpha-solanine. The plant sterols beta-sitosterol and fucosterol showed higher affinity for glycoalkaloids as compared to cholesterol and ergosterol. The mode of action of the glycoalkaloids is proposed to consist of three main steps: (1) Insertion of the aglycone part in the bilayer. (2) Complex formation of the glycoalkaloid with the sterols present. (3) Rearrangement of the membrane caused by the formation of a network of sterol-glycoalkaloid complexes resulting in a transient disruption of the bilayer during which leakage occurs.

Cytotoxicity of S-(1,2,3,4,4-pentachlorobutadienyl)-L cysteine after apical and basolateral exposure of LLC-PK monolayers. Involvement of an amino acid transport system.

Glutathione conjugation and subsequent formation of cysteine conjugates are key steps in the nephrotoxicity of halogenated alkenes. In this metabolic activation several organs are involved. However little is known about the transporters responsible for the uptake of cysteine conjugates. Recent evidence suggest that amino acid transporters play a role in this uptake. Monolayers of LLC-PK1 cells, a kidney cell line, were exposed to S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-CYS). Cytotoxicity was used as a parameter for PCBD-CYS uptake. Basolateral exposure (1 h: 400 microM and 16 h: 25 microM) to PCBD-CYS resulted in a much higher aminooxyacetic acid inhibitable cytotoxicity than apical exposure, suggesting a preferential basolateral uptake of PCBD-CYS. Exposure to PCBD-CYS in the absence of sodium did not result in a decrease of the cytotoxicity, suggesting a sodium independency of the PCBD-CYS uptake. Amino acids and amino acid analogues were used as diagnostic compounds in the further identification of the PCBD-CYS transporter. In cis-inhibition experiments monolayers were co-incubated with PCBD-CYS and these diagnostic compounds during one hour. System L substrates such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) and cycloleucine did not inhibit cytotoxicity. D-Tryptophan, a model inhibitor of System T, caused a strong inhibition. System L has, in contrast to System T, a high sensitivity to trans-stimulation. Pre-loading the monolayers with the diagnostic compounds should cause an increase in cytotoxicity when System L is involved. Neither System L substrates such as BCH and cycloleucine nor D-tryptophan increased cytotoxicity. These results suggest a preferential basolateral uptake of PCBD-CYS in LLC-PK1 monolayers and involvement of an amino acid transporter with characteristics of System T.