Scott D. Hauser

Selective inhibition of cyclooxygenase (COX)-2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis.

Prostaglandins formed by the cyclooxygenase (COX) enzymes are important mediators of inflammation in arthritis. The contribution of the inducible COX-2 enzyme to inflammation in rat adjuvant arthritis was evaluated by characterization of COX-2 expression in normal and arthritic paws and by pharmacological inhibition of COX-2 activity. The injection of adjuvant induced a marked edema of the hind footpads with coincident local production of PGE2. PG production was associated with upregulation of COX-2 mRNA and protein in the affected paws. In contrast, the level of COX-1 mRNA was unaffected by adjuvant injection. TNF-alpha and IL-6 mRNAs were also increased in the inflamed paws as was IL-6 protein in the serum. Therapeutic administration of a selective COX-2 inhibitor, SC-58125, rapidly reversed paw edema and reduced the level of PGE2 in paw tissue to baseline. Interestingly, treatment with the COX-2 inhibitor also reduced the expression of COX-2 mRNA and protein in the paw. Serum IL-6 and paw IL-6 mRNA levels were also reduced to near normal levels by SC-58125. Furthermore, inhibition of COX-2 resulted in a reduction of the inflammatory cell infiltrate and decreased inflammation of the synovium. Notably, the antiinflammatory effects of SC-58125 were indistinguishable from the effects observed for indomethacin. These results suggest that COX-2 plays a prominent role in the inflammation associated with adjuvant arthritis and that COX-2 derived PGs upregulate COX-2 and IL-6 expression at inflammatory sites.

Cloning and in vivo expression of bovine growth hormone receptor mRNA.

A cDNA for the bovine growth hormone (bGH) receptor has been cloned out of a cDNA library prepared from liver of a pregnant Holstein heifer. The cDNA clone hybridizes to a single 4.5 kb mRNA species and shares a high degree of sequence homology with growth hormone receptors cloned from other species. Utilizing the bGH receptor cDNA as a probe, a relatively high level of bGH-receptor mRNA was detected in bovine liver. In comparison to liver values, lower concentrations of bGH-receptor mRNA were detected in bovine kidney, anterior pituitary, and mammary gland. Because specific binding sites for bGH have not been convincingly demonstrated in isolated cell membranes from whole bovine mammary tissue, mammary tissue from two pregnant heifers (separate experiments) was separated into fractions enriched for epithelium, stroma, and blood components. These fractions were then probed for growth hormone receptor mRNA using solution hybridization-nuclease protection assays performed on isolated RNA. The assay results indicated that a low level of bGH-receptor mRNA is relatively evenly distributed throughout the mammary tissues of the two cows studied. In contrast, experiments using a probe to bovine insulin-like growth factor-I (IGF-I) indicate that the IGF-I mRNA is localized in the stromal/blood component of the mammary gland. These data suggest a possible paracrine mechanism for bGH action in the mammary gland.

Variants of somatotropin in cattle: gene frequencies in major dairy breeds and associated milk production

The amino acid sequence of bovine somatotropin (bST) varies at position 127 where either valine or leucine is found. The frequencies of leucine127 and valine127 bST gene alleles in cows (n = 302) and sires (n = 70) from major dairy breeds (Holstein, Brown Swiss, Guernsey, Jersey, and Ayrshire) were determined using DNA extracted from whole blood or spermatozoa. A 428 base pair fragment of the bST gene was amplified using polymerase chain reaction (PCR) and variants of the bST gene were detected as polymorphisms by Alu I restriction endonuclease digestion of PCR products. Restriction enzyme DNA fragments for the leucine127 variant were 265, 96, 51, and 16 base pair and for the valine127 variant were 265, 147, and 16 base pair as a polymorphism of bST was present in the 147 base pair DNA fragment. Frequencies of leucine127 and valine127 alleles for cows (n = 302) were 1.0 and 0 for Brown Swiss, .93 and .07 for Holstein, .92 and .08 for Guernsey, .79 and .21 for Ayrshire, and .56 and .44 for Jersey, respectively. In Holstein sires used for artificial insemination (n = 70), the frequency of leucine127 and valine127 alleles was .96 and .04. Estimates of transmitting ability for milk production tended to be greater for Holstein cows that were homozygous for leucine127 bST and Jersey cows that were homozygous for valine127 bST whereas Holstein sires with different bST genotypes were similar. In summary, frequencies of alleles for the bST gene were not similar in different dairy breeds and estimates of milk production were correlated with bST gene variant in cows but not sires.

Regulation of prostaglandin biosynthesis in vivo by glutathione.

Intraperitoneal administration of urate crystals to mice reduced subsequent macrophage conversion of arachidonic acid (AA) to prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid for up to 6 h. In contrast, levels of 12-hydroxyheptadecatrienoic acid (12-HHT) were markedly elevated. This metabolic profile was previously observed in vitro when recombinant cyclooxygenase (COX) enzymes were incubated with reduced glutathione (GSH). Analysis of peritoneal GSH levels revealed a fivefold elevation after urate crystal administration. The GSH synthesis inhibitorl-buthionine-[ S, R]-sulfoximine partially reversed the urate crystal effect on both GSH elevation and PG synthesis. Moreover, addition of exogenous GSH to isolated peritoneal macrophages shifted AA metabolism from PGs to 12-HHT. Urate crystal administration reduced COX-1, but induced COX-2 expression in peritoneal cells. The reduction of COX-1 may contribute to the attenuation of PG synthesis after 1 and 2 h, but PG synthesis remained inhibited up to 6 h, when COX-2 levels were high. Overall, our results indicate that elevated GSH levels inhibit PG production in this model and provide in vivo evidence for the role of GSH in the regulation of PG biosynthesis.Intraperitoneal administration of urate crystals to mice reduced subsequent macrophage conversion of arachidonic acid (AA) to prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid for up to 6 h. In contrast, levels of 12-hydroxyheptadecatrienoic acid (12-HHT) were markedly elevated. This metabolic profile was previously observed in vitro when recombinant cyclooxygenase (COX) enzymes were incubated with reduced glutathione (GSH). Analysis of peritoneal GSH levels revealed a fivefold elevation after urate crystal administration. The GSH synthesis inhibitor L-buthionine-[S,R]-sulfoximine partially reversed the urate crystal effect on both GSH elevation and PG synthesis. Moreover, addition of exogenous GSH to isolated peritoneal macrophages shifted AA metabolism from PGs to 12-HHT. Urate crystal administration reduced COX-1, but induced COX-2 expression in peritoneal cells. The reduction of COX-1 may contribute to the attenuation of PG synthesis after 1 and 2 h, but PG synthesis remained inhibited up to 6 h, when COX-2 levels were high. Overall, our results indicate that elevated GSH levels inhibit PG production in this model and provide in vivo evidence for the role of GSH in the regulation of PG biosynthesis.