The N-acetyltransferase (NAT) phenotype is an important determinant of individual susceptibility to occupational bladder cancer. N-Acetyltransferases arc known to metabolize aromatic amine bladder carcinogens, but the functional significance of NAT expression in the target organ is unclear. To resolve this issue, polygonal antisera against purified recombinant enzymes and C-terminal peptides of human NAT Type 1 (NAT1) and Type 2 (NAT2) were generated. Western blot analysis of exfoliated cells from human urine, pig bladder homogenate, and human bladder tumor-derived cell lines showed that NAT1 was expressed in all three systems, whereas NAT2 did not appear to be expressed in the bladder. Immunohistochemical analysis of human bladder tumor sections indicated that well-differentiated tumor cells expressed NAT1, with the highest level of expression being found in the umbrella cells that line the bladder lumen. Poorly differentiated tumor regions appeared to express NAT1 at lower levels than did well-differentiated areas. These findings support the hypothesis that aromatic amines are metabolized in the bladder epithelium by NAT1.

Primary cultures of human tracheal epithelial (HTE) cells cultured in vitro, in defined serum-free media, express prostaglandin endoperoxide G/H synthase (PGHS) activity and produce prostaglandin E2 (PGE2). In contrast to every other cell type studied to date, HTE cells appear to constitutively express PGHS-2, the ‘inducible’ form of the enzyme, while expressing little or no PGHS-1, the ‘housekeeping’ isoenzyme in vitro. Prostaglandin synthesis in HTE cells was reduced by a selective PGHS-2 inhibitor, N-(2-cyclohexyloyl-4-nitrophenyl] methane-sulfonamide (NS398), with an IC50 of approximately 1 μM. Immunoblotting and immunoprecipitation of enzymatic activity with isozyme-specific antisera revealed only the PGHS-2 isoform. Full length human cDNA probes detected only PGHS-2 message in Northern blots. Neither PGHS-2 activity nor mRNA levels were dependent on, nor stimulated by peptide growth factors present in the defined serum-free growth medium, or by serum. Prolonged maintenance in the absence of retinoic acid, however, lead to a decline in PGHS activity. Phorbol-myristate acetate (PMA) induced PGHS-2 activity and mRNA and neither PMA-induced, nor constitutive PGHS-2 expression was suppressed by corticosteroids. Actinomycin D-treatment for six hours reduced the PGHS-2 activity and mRNA to only 50% that of untreated cells, suggesting that PGHS-2 mRNA is extremely stable in these cells. HTE cells, at least in vitro, appear unique among prostaglandin-producing cells in that they express PGHS-2, constitutively, independent of regulation by growth factors, serum, or corticosteroids and fail to express PGHS-1 under any culture condition studied.

The mitogen-activated protein kinase (MAPK) signalling pathway serves to translocate information from activated plasmamembrane receptors to initiate nuclear transcriptional events. This cascade has recently been subdivided into two analogous pathways: the extracellular signal-regulated kinase (ERK) cascade, which preferentially signals mitogenesis, and the stress-activated protein kinase (SAPK) cascade, which is linked to growth arrest and/or cellular inflammation. In concurrent experiments utilizing rat glomerular mesangial cells (MCs), we demonstrate that growth factors or sphingosine activate ERK but not SAPK. In contrast, inflammatory cytokines or cell-permeable ceramide analogues activate SAPK but not ERK. Ceramide, but not sphingosine, induces interleukin-6 secretion, a marker of an inflamed phenotype. Moreover, ceramide can suppress growth factor- or sphingosine-induced ERK activation as well as proliferation. These studies implicate sphingolipid metabolites as opposing regulators of cell proliferation and inflammation through activation of separate kinase cascades.

Vasoconstrictor peptides, such as angiotensin II (Ang II) and endothelin-1 (ET-1), stimulate the synthesis and release of vasodilatory prostaglandins from multiple tissues and diverse cellular types. The synthesis of PGE2 and PGI2 acts as a negative feedback loop to antagonize the contractile actions of Ang II and ET-1. Inhibition of prostaglandin synthesis with nonsteroidal anti-inflammatory drugs (NSAIDs) enhances the constrictor actions of Ang II and ET-1 on the vasculature of the kidney and on the glomerulus. The enhanced production of prostaglandins, in response to constrictor peptides, is both short- and long-term. Prostaglandin synthesis is regulated at multiple steps, including: (1) phospholipase A2, which releases arachidonic acid from membrane phospholipids; (2) PGH synthase (PGHS), which converts arachidonic acid to the endoperoxides PGG2 and PGH2; and (3) PG synthases convert the endoperoxides to PGI2, PGE2, and others. ET-1 acutely activates phospholipase A2 through phosphorylation and acute increases of intracellular calcium. ET-1 also chronically enhances phospholipase A2 activity by transcriptional induction of this enzyme. There are no known acute effects of ET-1 to acutely enhance PGHS activity through posttranslational modification of the molecule. Chronically, however, cellular content of PGHS-2 can be induced through transcriptional induction of the enzyme in response to ET-1. Hence, ET-1 short- and long-term activates a modulatory feedback pathway that depends on upregulation of arachidonic acid release through phospholipase A2 and enhanced synthesis of prostaglandin endoperoxides through PGHS-2.

Endothelin-1 stimulates vascular smooth muscle and mesangial cells to release prostaglandin E2 which attenuates the vasoconstrictor and mitogenic effects of endothelin. The role of endothelin-1 to regulate prostaglandin endoperoxide synthase (PGHS)-1 and -2 gene expression and protein synthesis was evaluated in cultured mesangial cells. Endothelin induced mRNA and protein expression for PGHS-2 but not for PGHS-1. A direct correlation was observed between the mass of immunoprecipitated PGHS protein and PGE2 synthetic enzymatic activity.

Sphingosine is a product of sphingolipid metabolism that has been linked to a protein kinase C-independent mitogenic response. In previously published data, utilizing an in vitro model system for platelet-derived growth factor (PDGF)-induced vascular smooth muscle proliferation, we have demonstrated that sphingosine is increased at the expense of a concomitant decrease in ceramide formation, implicating an altered ceramidase activity. To explore mechanisms of growth factor-stimulated sphingosine formation, we have developed and investigated a cell-free model system assessing ceramidase activity. We now report that an alkaline, membrane-associated, ceramidase activity in the rat glomerular mesangial cell, a smooth muscle-like pericyte, is up-regulated by growth factors, apparently via a tyrosine kinase phosphorylation mechanism. PDGF also stimulated sphingomyelinase activity which generates sufficient substrate to drive the subsequent ceramidase reaction. Inflammatory cytokines, including interleukin-1, and tumor necrosis factor-alpha, stimulated sphingomyelinase but not ceramidase activity, a result consistent with the cellular accumulation of the ceramide, apoptidic, differentiating second messenger. Mitogenic vasoconstrictor peptides such as endothelin-1 stimulated neither sphingomyelinase nor ceramidase activities. An inhibitor of ceramidase activity, N-oleoylethanolamine, reduced PDGF- but not endothelin-1-stimulated proliferation. Thus, we conclude that, in mesangial cells, growth factors but not vasoconstrictor peptides or cytokines induce mitogenesis, in part, through ceramidase-mediated sphingosine formation.

We evaluated the role of endothelin-1 (ET-1), a vasoconstrictor peptide, to regulate prostaglandin endoperoxide synthase (PGHS)-1 and -2 gene expression and protein synthesis in cultured rat mesangial cells (MC). ET-1 induced mRNA for PGHS-2 but not PGHS-1 and also stimulated protein accumulation of PGHS-2 but not PGHS-1 in MC. ET-1 induction of PGHS-2 protein was accompanied by a sustained enhancement of enzymatic activity assessed by conversion of arachidonic acid to prostaglandin E2. The ET-1-stimulated PGHS-2 expression was reduced with protein tyrosine kinase but not with protein kinase C inhibitors or in protein kinase C-depleted cells. Both dexamethasone and heparin reduced ET-1-activated PGHS-2 mRNA expression and protein formation. We conclude that in MC, ET-1 induces PGHS-2 through a protein tyrosine kinase-dependent pathway.

Many arylamine and hydrazine drugs and xenobiotics are acetylated by liver N-acetyltransferase (NAT; EC 2.3.1.5). Two loci, mnat and pnat, encode the enzymes designated monomorphic and polymorphic NAT (mNAT and pNAT) respectively. These isoenzymes have different substrate specificities. In addition, at the polymorphic locus a diversity of alleles is found, which differ by specific point mutations that may or may not result in amino acid substitutions. These point mutations result in the 'slow' acetylation of substrates of pNAT. The substrates for NAT include carcinogenic arylamines. Susceptibility to bladder cancer has been related to slow acetylation. NAT has been characterized in immortalized human cell lines to assess their use in studies of the metabolism or arylamines in vitro. A monocytic cell line (U937) and two hepatoma cell lines of parenchymal lineage (HepG2 and Hep3B) have been shown to catalyse acetylation of substrates of mNAT but do not acetylate sulphamethazine, a substrate specific for pNAT. Using PCR to amplify the alleles of pNAT, followed by restriction-enzyme digestion of the product, the cell lines have been genotyped: U937 cells are homozygous slow acetylators (S1a/S1a) and HepG2 cells are heterozygous slow acetylators (S1a/S2). Transcription of pnat was confirmed in the hepatoma cell lines, by amplification of cDNA generated from these cells. In addition, splicing of mRNA specific for pNAT has been demonstrated by using a primer which anneals to a region in the 5' promoter region. Unlike the hepatoma cell lines, in U937 cells the pNAT gene is not transcribed. However, transcription of mnat was shown to occur in all three cell lines.

The effects of low level exposure of rats to 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) on their immune system was investigated. Dietary administration to young adult male Leeds strain rats of a total dose of 3 μg/kg body weight of TCDD resulted in an exposure duration-dependent reduction of in vitro lipopolysaccharide-induced production of interleukin (IL)-1 in cultures of their splenic macrophages. A 30-day exposure produced approximately 30% suppression and 180-day exposure produced approximately 52% suppression. This reduction did not negatively influence lipopolysaccharide-induced proliferation of B cells, instead an enhancement of B cell proliferation was observed after 30 days exposure. A 180 day exposure significantly suppressed the generation of IL-2 by either concanavalin A or phorbol myristate acetate/calcium ionophore stimulation, and reduced the lectin-induced proliferation of splenic T cells. The 30-day TCDD exposure showed no such immunotoxicity. TCDD at both exposure durations suppressed the expression of the α chain of the IL-2 receptor in concanavalin A-activated T cells, without affecting the CD4+/CD8+ ratio. The results suggest that exposure to a low dietary dose of TCDD suppresses the functions of several T cell subsets, some of the immunotoxic effects being produced early, while others require a longer exposure period. The effect of TCDD on B cells appears to be of transient nature, with less potentially serious consequences. Such exposure also down-regulates the IL-1 production function of macrophages. A common mechanism of TCDD immunotoxicity may be on the multifunctional signal transduction pathways downstream to the activation of protein kinase C and Ca2+ flux.

Many arylamine and hydrazine drugs and xenobiotics are acetylated by N-acetyltransferase (NAT), a cytosolic enzymic activity which has a wide tissue distribution. Humans can be classified as either fast or slow acetylators on the basis of their ability to metabolise isoniazid or sulphamethazine. These are termed polymorphic substrates. The acetylation of other compounds does not vary amongst individuals, e.g., p-aminobenzoic acid, and are termed monomorphic substrates. NAT from human hepatic and non-hepatic tissues, viz., (i) liver, (ii) the hepatoma cell line HepG2, (iii) tonsil lymphocytes and (iv) the monocytic cell line U937 have been compared with respect to substrate specificity towards polymorphic and monomorphic substrates. The chromatographic and centrifugation behaviour of NAT from these sources has also been investigated. NAT from liver shows 2-fold greater activity towards sulphamethazine than towards p-aminobenzoic acid as substrate. All other cell types tested show at least 70-fold greater activity with p-aminobenzoic as substrate compared to sulphamethazine. NAT from HepG2 cells, U937 cells and tonsil lymphocytes migrates as a single peak during ion-exchange chromatography, whereas the liver NAT activity is separated into two peaks. NAT in HepG2 cells resembles extra-hepatic tissue NAT rather than NAT in liver. HepG2 cells do not therefore represent a good in vitro model for investigation of human metabolism of arylamines or hydrazines. The molecular weight of NAT from U937 cells has been determined by a combination of sucrose density gradient centrifugation and gel filtration to be 31600 +/- 1200 daltons.