Ikuo Morita

A Novel Method for Prostaglandin Endoperoxide H Synthase Activity in Individual Intact Cells

Prostaglandins are key mediators of inflammation1. Prostaglandin endoperoxide H is synthesized by two related prostaglandin endoperoxide H synthases (PGHS-1 and PGHS-2, COX-1 and COX-2). PGHS-1 is a constitutive enzyme present in many but not all mammalian cells2. PGHS-2 is undetectable in most mammalian tissues, but expression of this isozyme can be induced by cytokines, growth factors, and tumor promoters3. This means that PGHS-2 will be closely related with inflammation. Therefore, a development of nonsteroidal anti-inflammatory drugs (NSAIDs) which inhibit PGHS-2 specifically is undergoing in the world. In the present paper, we will show a novel and convenient method for PGHS activity in intact individual cells.

Differential Effects of Nitric Oxide on Cyclooxygenase 1 and 2 Activities in Cultured Cells

Nitric oxide (NO) is an endogenous free radical mediator contributing to vascular homeostasis and neuronal plasticity. NO activates guanylate cyclase by binding tightly to heme Fe2+, which causes the formation of nitrosyl heme. As a result, an allosteric conformational change of the apo-enzyme is induced that activates the enzyme. Because cyclooxygenase (COX) is also a heme protein, there is a possibility that NO could affect prostaglandin (PG) synthesis by a similar mechanism. However, the role of NO in PG synthesis is still considered controversial. Salvemini et al. [1] reported that NO enhanced PGE2 production in hydronephrotic kidney. On the other hand, Tsai et al. [2] did not find any effect of NO on PG synthesis using purified cyclooxygenase. However, Kelner et al. [3] demonstrated that NO induced PGH2/PGE2 isomerase and enhanced PGE2 production in NIH 3T3 cells. To explore this further, we did the following experiments in cultured cells.

Collagen synthesizing activity in myofibroblasts isolated from scar tissue in rat oral mucoperiosteum.

Myofibroblasts (MFb) were isolated from scar tissue in rat oral mucoperiosteum and cultured in vitro. Collagen synthesizing activity in the MFb was compared with that in fibroblasts (Fb) derived from normal tissue. In the present study, collagen synthesizing activity in MFb was higher than that in Fb. On the other hand, no significant difference was found in non-collagen protein synthesizing activity between MFb and Fb. As a result, the ratio of the synthesizing activity of collagen to that of total protein was higher in MFb than Fb. Collagen type analysis showed that Type III/Type I ratio was higher in MFb than in Fb.

Change of arachidonic acid metabolism in inflamed rat dental pulp.

The profile of arachidonic acid metabolism in rat dental pulp was studied with a cell-free system. Conversion of exogenously added radioactive arachidonic acid was measured by using thin-layer chromatography. Normal dental pulp could mainly produce 6-keto-PGF1α and 12- or 15-HETE like lipoxygenase product. When rat dental pulp was experimentally inflamed by applying bacterial lipopolysaccharide, increase of arachidonic acid metabolizing activity was observed. Especially PGE2 producing activity was elevated 9.3-fold compared with that of the normal. It was suggested that arachidonic acid metabolites were involved in the development of pulpal inflammation.