Both cyclooxygenase-1 and -2 are expressed in the spinal cord, and the spinal COX product prostaglandin E(2) (PGE(2)) contributes to the generation of central sensitization upon peripheral inflammation. Vice versa spinal COX inhibition is considered an important mechanism of antihyperalgesic pain treatment. Recently, however, COX-2 was shown to be also involved in the metabolism of endocannabinoids. Because endocannabinoids can have analgesic actions it is conceivable that inhibition of spinal COX produces analgesia not only by inhibition of PG synthesis but also by inhibition of endocannabinoid breakdown. In the present study, we recorded from spinal cord neurons with input from the inflamed knee joint and we measured the spinal release of PGE(2) and the endocannabinoid 2-arachidonoyl glycerol (2-AG) in vivo, using the same stimulation procedures. COX inhibitors were applied spinally. Selective COX-1, selective COX-2 and non-selective COX inhibitors attenuated the generation of spinal hyperexcitability when applied before and during development of inflammation but, when inflammation and spinal hyperexcitability were established, only selective COX-2 inhibitors reversed spinal hyperexcitability. During established inflammation all COX inhibitors reduced release of spinal PGE(2) almost equally but only the COX-2 inhibitor prevented breakdown of 2-AG. The reversal of spinal hyperexcitability by COX-2 inhibitors was prevented or partially reversed by AM-251, an antagonist at the cannabinoid-1 receptor. We conclude that inhibition of spinal COX-2 not only reduces PG production but also endocannabinoid breakdown and provide evidence that reversal of inflammation-evoked spinal hyperexcitability by COX-2 inhibitors is more related to endocannabinoidergic mechanisms than to inhibition of spinal PG synthesis.
A versatile, multidimensional, and non-denaturing proteome separation procedure using microplate technology is presented, yielding a digitized image of proteome composition. In the first dimension, the sample under study is separated into 96 fractions by size exclusion chromatography (SEC). In the second dimension, the fractions of the first dimension are transferred by the liquid-handling device CyBi-Well (CyBio AG, Jena, Germany) to 96 parallel anion exchange chromatography columns. In this way the proteins are conserved in their native states and are distributed in 2400 liquid fractions with high recovery rates and sufficient reproducibility. The resulting fractions are subjected to protein quantitation and identification. Spectrophotometrical and immunological methods and enzyme activity measurements are used for quantitation. To identify proteins, the fractions are subjected to MALDI-MS, and their tryptic digests to both MALDI- and LC-ESI-MS/MS. All preparation steps except the first are applied in parallel to sets of multiples of 96 samples. The procedure may be refined by adding more separation steps and may be adapted to various protein amounts and to various proteomes. Moreover, the method offers the opportunity to investigate functional protein complexes. The method was applied to separate the normal human serum proteome. Within 255 fractions exhibiting the highest protein concentrations, 742 proteins were identified by LC-ESI-MS/MS peptide sequence tags.
Cathepsin S, a lysosomal cysteine protease, is synthesized as inactive precursor. It is activated in the lysosomes by a proteolytic cleavage of the propeptide. HEK 293-cells which do not express cathepsin S were transfected with cDNA of either wild type human procathepsin S or a mutant procathepsin S in which Asn of the only glycosylation site in the proregion was replaced by Gln. The cells expressed glycosylated and non-glycosylated procathepsin S, respectively. Large amounts of the precursors were secreted into the culture media by both transfectants. Secreted wild type procathepsin S contained Man-6-phosphate in the oligosaccharide chain. Wild type procathepsin S was activated in the cells but no maturation occurred in the culture media. In vitro processing of glycosylated as well as of non-glycosylated procathepsin S gave fully active enzymes thus indicating that the oligosaccharide chain was not necessary for proper folding. A reuptake of the glycosylated and non-glycosylated procathepsin S by HEK 293-cells could be observed. Small amounts of mature cathepsin S were detected in the lysosomes of the mutant transfectants. Subcellular fractionation showed non-glycosylated procathepsin S in the membrane fraction. Non-glycosylated procathepsin S was bound to the plasma membrane at 2 degrees C, suggesting an additional sorting motif in the cathepsin S molecule besides the Man-6-phosphate residue.
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