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Cyclic AMP and cyclic GMP, considered to be cell proliferation regulators, have been reported to fluctuate in proliferating fibroblasts in vitro. The objectives of this experiment were to study the localization, distribution and staining patterns of these cyclic nucleotides in mechanically-stressed, proliferating periodontal fibroblasts in vivo. Cat canines were tipped by force applications for 0 to 48 hours and serial sagittal sections of fresh frozen, unfixed, undecalcified jaws were processed immuno-histochemically for the localization of cAMP and cGMP. Periodontal tension sites were studied microscopically. Fibroblastic staining for cAMP, which was localized mainly in the cell periphery, did not change appreciably as a result of tension. However, staining intensity increased one hour after the application of force, decreased after 6 hours and increased again at 24 hours. Staining for cGMP, initially covering the entire cell area, was concentrated over nuclei three hours after onset of tension, and diffused over cell periphery and cytoplasm thereafter. Intensity of staining for cGMP was maximal at 3 hours and low at 12 and 48 hours. These results demonstrate that stress-induced fibroblastic responses in vivo involve alterations in staining intensity for both cyclic nucleotides which may correspond with fluctuations of these regulators, reported to occur in vitro in various stages of the mitotic cycle. Moreover, this technique enables the identification of the involved cells in a stimulated, non-synchronized cell population.

Technetium-99-methylene diphosphonate (99mTc-MDP) bone images were obtained from live cats whose mandibles had been stimulated non-invasively and unilaterally with electric current (12 +/- 2 microA) for 1 week. The images were obtained using a gamma camera interfaced to a digital computer for quantitative comparison of the mandible. Radionuclide uptake by the stimulated sides increased by 59 +/- 26 per cent when compared with the contralateral control sites. Immunohistochemical staining of mandibular sections for adenosine 3',5'-monophosphate (cyclic AMP) showed that periosteal osteoblasts opposite the electrodes were intensely stained. Thus 99mTc-MDP scintigraphy is a sensitive non-invasive technique for determining in vivo enhanced bone remodelling activity. The immunohistochemistry indicates that the stimulation of bone cells was limited to surfaces adjacent to both cathode and anode.

Previous experiments indicate that bone cells respond to external stimuli with fluctuations of cyclic nucleotide levels. The objective of this experiment was to study the response of alveolar bone to the application of tensile forces through an examination of the osteoblastic staining pattern for cAMP and cGMP. Cat canines were tipped by 80-g force for 0 to 48 hours. Fresh frozen, unfixed, undecalcified jaws were sectioned sagittally and stained immuno-histochemically for cAMP and cGMP. In tension sites, osteoblastic staining intensity for cAMP decreased gradually from one to three hours, and then increased by 24 hours. Intense staining for cGMP, visible in osteoblasts of all treated cats, peaked after three hours of treatment and then again at 24 hours. Generally, groups of cGMP-stained osteoblasts were found adjacent to unstained osteoblasts. The observed fluctuations in the osteoblasts staining pattern for cAMP and cGMP indicates involvement of these substances in the early response of osteoblasts to mechanical stimuli in vivo.

The cyclic AMP and cyclic GMP concentrations of alveolar bone of control and PTE-treated cats were measured by chemical and immunohistochemical methods. In the PTE-treated animals, alveolar bone osteoblasts stained intensely for cAMP, but very weakly for cGMP; the periodontal ligament (PDL) cells stained for cAMP similarly to the controls, but some PDL cells stained more intensely for cGMP than their controls; osteocytes stained for cAMP with greater intensity than in the controls; osteoclasts stained intensely for both cyclic nucleotides. We found that bone samples taken from animals 20 and 60 min after administration of PTE contained twice the amount of cAMP, and almost three times the amount of cGMP observed in the controls. These results indicate that the cellular source of bone cyclic nucleotides in PTE-treated animals varies as to cell type, and therefore in bone and PDL the functions mediated by cAMP are not necessarily antagonistic to those mediated by cGMP.

During development of the mammalian secondary palate, medial-edge epithelia (MEE) from apposing palatal shelves adhere and undergo autolysis allowing palatal mesenchymal regions to unite. In a prior study (Greene & Pratt, 1979), we reported a transient increase in levels of cyclic AMP (cAMP) in the mouse and rat palate during epithelial adhesion and cell death. The objective of this study was to examine the distribution of cyclic AMP in the developing rodent secondary palate using immunohistochemistry to localize cyclic AMP. Staining for cAMP was observed in the epithelium just prior to and during epithelial fusion (day 16 in the rat; day 14 in the mouse). Cyclic AMP was distributed throughout the epithelial cytoplasm whereas no staining was seen in nuclei. Epithelial staining for cAMP was faint or absent 24 and 48 h prior to epithelial contact. Mesenchymal staining for cAMP was minimal and associated with the plasma membrane at all stages studied. These results demonstrate that elevated levels of cAMP in the rat and mouse palate during epithelial adhesion and cell death are mainly due to increases of the nucleotide in palatal epithelium. This observation suggests that a transient increase in epithelial cAMP may play a role in palatal epithelial differentiation.

Cyclic adenosine 3',5'-monophosphate (cAMP) concentrations and cellular distribution were studied in dental and periodontal tissues during tooth eruption in kittens. Although the mean levels of cAMP around developing teeth were similar in all the tissue samples, there were marked differences in cAMP stainability of tissues apical and occlusal to the erupting teeth.

Previous experiments indicate that bone cells respond to externally applied stimuli with fluctuations of cyclic nucleotide levels. The objective of this experiment was to study the response of bone at the midpalatal suture to the application of tensile forces in young and old animals, through an examination of the osteoblastic staining pattern for cAMP and cGMP. The midpalatal suture was rapidly expanded for 0, 10, and 15 days, respectively, in three young and three old cats. Fresh, frozen, unfixed, undecalcified maxillae were sectioned transversely and stained immunohistochemically for cAMP and cGMP. The staining intensity for both nucleotides was increased in the osteoblasts of the young treated animals, while in the old animals the osteoblasts were only faintly stained for cAMP and cGMP. These results demonstrate that bone cells of old animals are less responsive to tensile forces than the corresponding cells in young animals.

Cyclic nucleotides (cAMP and cGMP) and prostaglandin E (PGE) have been implicated as possible mediators of the effects of external stimuli on bone cells. The objective of this study was to determine changes in relative levels of these substances in mineralized tissue cells in response to mechanical and electrical stimuli, by the use of a combined immunohistochemical-microphotometric procedure. Canine teeth of eight 10-12 month-old female cats were tipped distally with 80 g force for either 1 h or 14 days. After 1 h, a slight elevation of staining intensity in alveolar bone osteoblasts and periodontal ligament (PDL) cells was observed at sites of tension and compression. After 14 days of treatment, this effect was markedly increased. Fifteen female cats, 10-12 months old, received electric stimulation (20 micron amperes d.c.) to the gingiva of 1 maxillary canine for 1, 5, 15, 30, or 60 min. At the cathode, significant increases of staining intensity in periosteal osteoblasts for cAMP, cGMP, and PGE were found at 15 and 60 min. At the anode, a significant rise in the staining intensity of these cells for PGE was seen at 15 min; at 60 min, cGMP and PGE, but not cAMP, were elevated. These results demonstrate the usefulness of the immunohistochemical technique in detecting relative changes in mineralized tissue cell content of cyclic nucleotides and prostaglandins in response to local application of physical stimuli of short and long duration.