Exposure to inflammatory agents or cytokines causes the suppression of cytochrome P450 (CYP) enzyme activities and expression in liver and primary hepatocyte cultures. We showed previously that phenobarbital-induced CYP2B protein is downregulated in primary cultures of rat hepatocytes after exposure to bacterial endotoxin (lipopolysaccharide) in a nitric oxide (NO) -dependent manner. In this study, we found that CYP2B proteins in primary rat hepatocyte cultures were suppressed >60% after 6 h of treatment with interleukin-1 (IL-1). This effect was NO-dependent, and treatment of cells with the NO donors (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione, and S-nitroso-N-acetylpenicillamine also suppressed CYP2B proteins. However, the down-regulation by IL-1 was insensitive to inhibition of cGMP-dependent protein kinases. Cytochrome P450s (CYP) 4 are products of multigene families, and many CYPs are involved in biosynthesis and catabolism of physiologically important active molecules such as steroid hormones, sterol, and fatty acids (1). One-third of them are involved in clearance of xenobiotic substances, most of which are expressed in the liver. Inflammation and infection cause the down-regulation of enzyme activity and expression of many CYPs leading to decreased drug clearance, elevation of plasma drug levels, and drug toxicity (2). Inflammation and infection can decrease the metabolic clearances of CYP substrates by 20 -70% (3).Substantial evidence exists to suggest a role of nitric oxide in the regulation of CYP enzyme activities in inflammation. NO is a free radical molecule with important regulatory roles in vasodilation, neurotransmission, inflammation, and cell signaling (4 -6). Inducible nitric-oxide synthase (NOS2, inducible NOS) is induced during infection and inflammation in vivo and in cell cultures treated with bacterial lipopolysaccharide (LPS) or cytokines (7), resulting in high levels of NO production in the cells. It is known that nitric oxide and NO donors are capable of inhibiting the catalytic activities of hepatic microsomal P450s (8 -14). For example, Chlamydia infection resulted in the reduction of CYP1A-and 2B-related metabolism by 49% in mouse liver (15), which was blocked by NOS inhibitors. Three mechanisms are suggested for inhibition of activity; 1) reversible ligation of NO to (primarily ferrous) P450 heme (8); 2) oxidation of P450 protein thiols (16); 3) nitration of specific tyrosine residues on the enzyme (17). CYP8A1 (prostacyclin synthase) and CYP2B1 have been shown to undergo tyrosine nitration by peroxynitrite in cell culture and in vitro, respectively (13,17).Although it is clear that NO production is not globally responsible for the down-regulation of CYP gene expression that occurs in inflammation and infection (18 -20), several reports suggested that inhibition of NOS can attenuate the down-regulation of some hepatic CYP mRNAs or proteins (21-23) or that NO donors can down-regulate some CYP mRNAs (24,25). This evidenc...
In transformed mouse fibroblasts, a significant proportion of the lysosomal cysteine protease cathepsin L remains in cells as an inactive precursor which associates with membranes by a mannose phosphate-independent interaction. When microsomes prepared from these cells were resolved on sucrose gradients, this procathepsin L was localized in dense vesicles distinct from those enriched for growth hormone, which is secreted constitutively when expressed in fibroblasts. Ultrastructural studies using antibodies directed against the propeptide to avoid detection of the mature enzyme in lysosomes revealed that the proenzyme was concentrated in dense cores within small vesicles and multivesicular endosomes which labeled with antibodies specific for CD63. Consistent with the resemblance of these cores to those of regulated secretory granules, secretion of procathepsin L from fibroblasts was modestly stimulated by phorbol, 12-myristate, 13-acetate. When protein synthesis was blocked with cycloheximide and lysosomal proteolysis inhibited with leupeptin, procathepsin L was found to gradually convert to the active single-chain protease. The data suggest that when synthesis levels are high, a portion of the procathepsin L is packaged in dense cores within multivesicular endosomes localized near the plasma membrane. Gradual activation of this proenzyme achieves targeting of the proenzyme to lysosomes by a mannose phosphate receptor-independent pathway.
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