To begin to answer these questions we have examined how drug trans port is altered in microglial cells following treatment with the prototypical inflammatory mediator lipopo lysacharride. We show that LPS exposure re duces cellular accumulation clearly of the protease inhibitor saquinavir and examine possible mechanisms under lying this effect. Methods and materials Materials Saquinavir was kindly provided from Roche Products Inc. saquinavir was syn thesized by Amersham. MK571 was obtained from Biomol. The monoclonal antibodies MRPr1 and C219 were purchased from Kamiya Biomedical, and Signet, respectively. 2 diazenolate 2 oxide was purchased from Alexis Biochemicals, bisindolylma leimide I, 1,4 Diamino 2,3 dicyano 1,4 bis butadiene, diphenyleneiodonium, endothelin 1, .

4 hydroxy 2,2,6,6 tetramethylpiperidene 1 oxyl, NG monomehtyl Inhibitors,Modulators,Libraries L arginine monoacetate, interleukin1beta, LPS . phorbol 12 myristate 13 acetate, 5 pregnen 3B ol 20 one 16 carbonitrile, prostaglandin E2, 1,9 pyrazoloanthrone, NF ��B inhibitor peptide, tissue inhibitor of metalloproteinase 3, tumor necrosis factor Inhibitors,Modulators,Libraries alpha, type I IL 1B receptor antagonist, wortmannin, and antibodies against IL1 B and TNF were all purchased from Calbiochem. Fucoidan was obtained from Sigma. All tissue culture reagents were purchased from Inhibitors,Modulators,Libraries Invitrogen unless otherwise indicated. Animals Timed pregnant Wistar and Fisher rats were purchased from Charles River Laboratories. Timed Pregnant, and C3HHeJ mice were purchased from The Jackson Laboratory. The C3HHeJ strain contains a spontaneous mutation in the TLR4 gene making these mice deficient in TLR4 mediated responses, where they are resistant to endotoxins such as LPS.

All animals were maintained in a strict pathogen free environment. All studies were approved by the National Institutes of En vironmental Health Sciences institutional review board and adhered to NIH guidelines for the care and handling of experimental animals. Primary cultures of microglia Primary microglia enriched cultures were prepared from whole brains Inhibitors,Modulators,Libraries of one to two day old mice and rats as de scribed previously, with modifications. Following decapitation, whole brains were removed, and brain tis sue triturated after meninges and blood vessel removal. Cells were seeded in complete medium were then obtained from shak ing the lightly adherent Inhibitors,Modulators,Libraries microglia, and seeding the cells in 24 well plates for subsequent assays.

Cells were used for subsequent experiments at 24 hours post shaking. Microglia cell line The continuous rat microglia cell line HAPI was origin ally isolated from mixed glial cultures prepared from three day old rat pups, and was a generous gift of Dr James R Connor. The cells exhibit prototypical microglia type behavior including the selleck kinase inhibitor ability to phagocytose, and to release TNF and NO upon stimulation with LPS.