Cytokines, interleukin-1 (IL-I), tumor necrosis factor a, and the neurotransmitter, substance P, have been implicated in the pathogenesis of arthritis because they stimulate synovial cells to secrete prostaglandin E, and collagenase in vitro. We investigated in vivo changes in intraarticular substance P and the degradation of cartilage proteoglycan in response to intraarticular cytokine injections in rabbits. Twenty-four hours after a single injection of 10 ng, 30 ng, or 100 ng of recombinant human &la (rHuIL-la) per joint, the mean f SEM levels of substance P detected in the cell-free joint lavage fluid were 250 & 67 fmoles, 480 2 60 fmoles, and 530 & 130 fmoles (n = 4-9, respectively. The level of substance P in the contralateral knees injected with diluent was 58 -c 8 fmoles (n = 12). The level of substance P had increased by 2 hours after IL-1 injection and remained elevated in the joint 48 hours after injection. Cytokineinduced proteoglycan depletion was also time-and dosedependent. Proteoglycan concentrations in articular cartilage dissected from the weight-bearing condyles were calculated as the ratio of sulfated glycosaminoglycan measured using 1,9-dimethyImethylene blue:hydroxyproline. After 48 hours, 10 ng, 30 ng, or 100 ng of rHuIL-la per joint decreased proteoglycan levels by 9 f 4%, 14 f 4%, and 21 2 3% (n = a), respectively. Likewise, the injection of recombinant human
Substance P, a neuropeptide mediator of inflammation, was quantified during the evolution of trinitrobenzene sulfonic acid (TNBS)-induced ileitis in guinea pigs. Ileitis was induced by a single intraluminal injection of TNBS (30 mg/kg in 50% ethanol). Misoprostol, a prostaglandin E1 analogue, was administered parenterally (30 mg/kg sc twice daily) in a group of TNBS-treated animals. Control guinea pigs received intraluminal saline (sham) or 50% ethanol (TNBS vehicle). Guinea pigs were evaluated at day 1, 3, 7, 14, or 30 after ileitis induction for substance P content (radioimmunoassay) and distribution (immunohistochemistry), morphology, myeloperoxidase (MPO) activity, and protein leak into the gut lumen. TNBS administration caused an increase in ileal MPO activity that peaked at day 7 and increased mucosal leak of protein. Misoprostol attenuated the granulocyte infiltration (MPO) response to TNBS but exacerbated the mucosal leak of protein. Substance P levels in whole ileal segments were unaltered from baseline on day 1 in all groups. On day 3 a marked decrease in ileal substance P content was evident in the TNBS and TNBS + misoprostol groups. As early as day 1, immunohistochemistry suggested that the decreased substance P content was confined to the mucosa and submucosa, because myenteric plexus staining was not reduced. Loss of staining in the perivascular nerves was particularly marked. Substance P content and distribution returned to baseline by day 30 post-TNBS, although MPO activity remained slightly elevated. We concluded that TNBS ileitis is associated with a marked reduction in mucosal and submucosal substance P content in parallel with the inflammatory response. Although misoprostol attenuated granulocyte infiltration in this model, it did not prevent the disturbances in enteric substance P or mucosal protein leak.
C-terminal amidation is a posttranslational modification found in many neuropeptides. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the synthesis of the biologically essential C-terminal amide from a glycine-extended precursor peptide. Reported herein are the first potent inhibitors of PAM. Dipeptides containing a C-terminal homocysteine and an N-acylated hydrophobic amino acid were found to inhibit PAM with IC50s in the low nanomolar range. Inhibition potency was dependent on both the carboxylate and the thiolate functionalities of the homocysteine and on the hydrophobic groups of the second amino acid. The thiolate was postulated to produce high binding affinities through coordination with the active-site copper. The compound series also exhibited potent inhibition of PAM in rat dorsal root ganglion cells as demonstrated by a dose-dependent increase in the substance P-Gly/substance P ratio. These results indicate that the compounds have sufficient potency and intracellular bioavailability to aid future studies focused on neuropeptide function and the contributions of neuropeptides to various disease processes.
Injection of 2.5 mg of lambda-carrageenan into the rat pleural cavity resulted in a time-dependent increase in pleural exudate substance P (SP) levels up to 24 hr. Synergistic increases in the exudate formation were observed when a sub-optimal quantity of carrageenan was injected with SP. Pre-treatment of rats with capsaicin at 50 and 100 mg/kg s.c. daily for one week prior to the induction of pleurisy blocked the increase in exudate volume and SP levels when compared to that normally detected after carrageenan injection. These results suggest that inhibition of SP production may improve inflammatory conditions.
The terminal step in the biosynthesis of substance P is the conversion of its glycine-extended precursor to the mature, amidated peptide by the alpha-amidating enzyme. This posttranslational modification was demonstrated in cultured, dissociated sensory neurons of dorsal root ganglia from neonatal rats. An assay was developed to quantitate both substance P and its precursor peptide in these cells. More than 90% of these two peptides was present as mature peptide in uncultured cells. In contrast, after 8 days in culture, about 85% of the peptides was the precursor, which increased 200-fold, whereas the level of substance P itself tripled during this culturing period. Since alpha-amidating enzyme requires ascorbate for activity, this reducing agent was added to the culture medium. Ascorbate induced a dose-dependent rise in the percentage of amidated peptide, with an apparent Km of 20 microM. After 5 days of culturing in the presence of 500 microM ascorbate, substance P increased 8-fold, constituting 70% of the total. The alpha-amidating enzyme also needs copper for activity. Even with 500 microM ascorbate in the culture medium, the copper chelator diethyldithiocarbamate dose-dependently reduced substance P synthesis by the sensory neurons, with a concomitant increase in its precursor peptide. These results suggest the presence of alpha-amidating enzyme in sensory neurons of dorsal root ganglia. It is likely that conversion of other glycine-extended precursors to their mature peptides in cell cultures would also require ascorbate and copper.
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