Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

Interactions between cyclosporin A, low-density lipoprotein and the low-density lipoprotein receptor

Cyclosporine A (CSA) is a cyclic eleven amino acid, lipophilic molecule used therapeutically as an immunosuppressive agent. Cyclosporine can specifically inhibit the transcription of a number of different genes. It is known that CSA is bound almost exclusively to lipoproteins in plasma, however, the relationship between the low density lipoprotein (LDL), the LDL receptor, and CSA has not been fully elucidated. The exact mechanism of cellular uptake of CSA is unknown, but it is believed to be by simple passive diffusion across the cell membrane. In addition, it has been recently shown that the frequent finding of hypercholesterolemia seen in patients treated with CSA can be explained by a CSA-induced effect. The mechanism by which CSA induces hypercholesterolemia is not known. We have used an LDL receptor-deficient animal model, the Watanabe Heritable Hyperlipidemic (WHHL) rabbit to investigate the role of LDL and the LDL receptor in the cellular uptake of CSA. Using this animal model, we have shown that CSA uptake by lymphocytes is predominantly LDL receptor-mediated. Chemical modification of apoB-100 on LDL particles abolishes their ability to bind to the LDL receptor. When CSA is incubated with modified LDL much less is taken-up than when native LDL is incubated with CSA. Treatment of two human cell lines with CSA results in a dose-dependent decrease in LDL receptor mRNA levels. Using a novel transfection system involving the 5$\sp\prime$-flanking region of the LDL receptor gene, we have found that CSA decreases the number of transcripts, but is dependent on whether or not cholesterol is present and the stage of growth of the cells. ^

Characterization of the interactions between two sites of plasticity engaged during eyelid conditioning

Here, we investigate the involvement of two sites of plasticity in the learning and expression of a simple associative motor behavior—the classically conditioned eyelid response. While previous studies clearly demonstrate that lesions of the anterior interpositus nucleus of the cerebellum abolish learned responses and prevent subsequent learning, studies investigating the effects of lesions of the cerebellar cortex on learning and retention have produced discrepant results. We complement ablative lesion studies of the cortex with the use of reversible, pharmacological blockade of cerebellar cortical transmission to investigate the role of the cerebellar cortex in eyelid conditioning. We demonstrate that both pharmacological blockade as well as focused ablative lesions of the cortex abolish timed responses and unmask responses with a fixed, short latency that are not displayed by the intact animal. Pharmacological blockade of cerebellar cortex output at various stages of acquisition and extinction reveals appropriate, learning dependent changes in the amplitude and probability of short latency responses during training. Acquisition of both short latency as well as timed responses is prevented by ablative lesions of the anterior lobe of the cerebellar cortex. These convergent results from technically distinct methods of removing the influence of the cerebellar cortex from conditioned behavior are consistent with the proposal that (1) eyelid conditioning engages two cerebellar sites of plasticity-one in the cortex and one in the anterior interpositus nucleus, (2) plasticity in the cerebellar cortex is necessary for proper response timing, (3) plasticity in the nucleus mediates the short latency responses unmasked by lesions of the cerebellar cortex, and (4) cerebellar cortical output is necessary for the induction of plasticity in the nucleus. ^