Immunohistochemical assessment of the effect of chemical and mechanical stimuli on cAMP and prostaglandin E levels in human gingival fibroblasts in vitro

Abstract These were evaluated by: (1) a combined immunohistochemical-microphotometric procedure (IH) and (2) conventional radiometric assays. Human gingival fibroblasts were in the sixth passage, grown and maintained in Dulbecco minimal essential medium (DMEM) supplemented with 10 per cent horse serum. For chemical and hormonal stimuli, cells (2 × 10 4 ) were seeded on tissue-culture chamber/slides, and incubated with graded doses of either parathyroid hormone (PTH) or prostaglandin E 2 (PGE 2 ) for assessment of their adenosine-3′,5′-monophosphate (cAMP) levels, and with indomethacin or colchicine for their effect on PGE levels. For mechanical stimuli, cells (1 × 10 6 ) were seeded on culture dishes with a flexible plastic membrane and stretched for 5, 30, 60 or 120 min by placing the membrane over a convex surface and weighting the dish cover. After freeze drying, cells were stained by an immunoperoxidase technique for either cAMP or PGE, using monoclonal antibodies. The staining intensity of fibroblasts was determined at 600 nm wavelength. Per cent light absorbance of 15 cells in each slide was measured and the results tested by analysis of variance. The gingival fibroblasts responded to the drugs and hormones in a dose- and time-related fashion. Stretching significantly increased their synthesis of PGE with concomitant increase in cAMP. The IH results were compared with the radiometric assays to confirm the validity of this technique; both assays were valid for describing the quantitative responses of these cells to the stimuli. In particular, the IH method could localize those intracellular sites which demonstrated chances in relative cAMP and PGE concentrations in response to hormonal stimuli.