Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules.

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.

Chronic multichannel recordings from organotypic hippocampal slice cultures: protection from excitotoxic effects of NMDA by non-competitive NMDA antagonists.

In vitro neuronal damage has traditionally been evaluated by biochemical or anatomical but not by electrophysiological techniques. In the present study, we combined two newly developed technologies, an 8 x 8 multi-electrode array (MED-64) and cultured hippocampal slices, to demonstrate the potential use of electrophysiological measures as index of neuronal damage. We first demonstrated the stability of electrophysiological recordings over prolonged periods of time (up to 14 days) in field CA1 of cultured hippocampal slices following electrical stimulation of the Schaffer collateral pathway. We then assessed the neurotoxic properties of NMDA and AMPA and determined that the time-course, potency, and efficacy of these two neurotoxins were similar to those assessed by other experimental approaches. We also compared the efficacy and potency of two non-competitive NMDA receptor antagonists to protect against NMDA-mediated neurotoxicity. Again, the results matched well with the results obtained from traditional techniques. Thus, this new technology might provide a new and powerful method to study the chronic effects of drugs or other experimental manipulations in an in vitro preparation.