Inflammation of the Human Dental Pulp Induces Phosphorylation of eNOS at Thr495 in Blood Vessels

Abstract

The activity of endothelial nitric oxide synthase (eNOS) in endothelial cells increased with the phosphorylation of the enzyme at Ser1177 and decreased at Thr495. The regulation of the phosphorylation sites of eNOS at Ser1177 and Thr495 in blood vessels of the healthy and inflamed human dental pulp is unknown. To investigate this, healthy and carious human third molars were immersion-fixed and decalcified. The localization of eNOS, Ser1177, and Thr495 in healthy and inflamed blood vessels was examined in consecutive cryo-sections using quantitative immunohistochemical methods. We found that the staining intensity of Ser1177 in healthy blood vessels decreased in inflamed blood vessels, whereas the weak staining intensity of Thr495 in healthy blood vessels strongly increased in inflamed blood vessels. In blood vessels of the healthy pulp, eNOS is active with phosphorylation of the enzyme at Ser1177. The phosphorylation of eNOS at Thr495 in inflamed blood vessels leads to a decrease in eNOS activity, contributing to eNOS uncoupling and giving evidence for a decrease in NO and an increase in O2− production. Since the formation of the tertiary dentin matrix depends on intact pulp circulation, eNOS uncoupling and phosphorylation of eNOS at Thr495 in the inflamed pulp blood vessels should be considered during caries therapy.

Dental Pulp Inflammation Initiates the Occurrence of Mast Cells Expressing the α1 and β1 Subunits of Soluble Guanylyl Cyclase

“…However, during inflammation, sGC is oxidized (from Fe 2+ to Fe 3+ ) by higher concentrations of O 2 − and ONOO − , leading to the insensitivity of sGC to NO [ 3 , 4 , 5 , 6 ]. Inflammation induces an increase in ROS and RNS concentrations [ 71 , 72 ]; this effect has been reported for inflamed dental pulp [ 49 , 50 ]. In addition, the inflammation-dependent synthesis of ROS and RNS in mast cells has also been described [ 70 ].…”

“…In inflammation, O 2 − and ONOO − oxidize sGC (Fe 2+ to Fe 3+ in the β 1 subunit of sGC), leading to the insensitivity of sGC to NO [ 3 , 4 , 5 , 6 ]. Because O 2 − and ONOO − have been detected at higher concentrations in inflamed dental pulp [ 49 , 50 ], we suggested that sGC might be present as an α 1 β 1 isoform in mast cells at the protein level and could be insensitive to NO in the oxidized state in these cells during the inflammation of human dental pulp.…”

“…Since the definitive diagnosis of pulpal inflammation is determined by histopathology, sections were stained with hematoxylin and eosin (HE) [ 49 ] and the results were characterized by histopathological diagnosis. In each subgroup of molars, immunohistochemical staining for mast cell tryptase (MCT), HLA-DR (monocytes, dendritic cells, and activated T and B cell markers), and CD68 (macrophage marker) were performed to characterize the leukocyte types in the healthy and inflamed human dentin–pulp complex and periodontium, as described previously [ 50 , 74 ].…”

“…The sections were incubated for 1 h with avidin–biotin–peroxidase complex (1:100) (Vector). The immunohistochemical reaction was developed in all incubations for 15 min with 0.05% 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO, USA) in 0.05 M Tris-HCl buffer, pH 7.6, containing 0.01% H 2 O 2 and 0.01% nickel sulfate [ 49 , 50 ].…”

“…To show the cell nuclei, sections were stained with DRAQ5 (Cell Signaling Technology, Frankfurt am Main, Germany) for 15 min. The sections were coverslipped with Aqua Poly/Mount (Polysciences Inc., Warrington, PA, USA) and analyzed with an LSM510 confocal microscope (Carl Zeiss, Jena, Germany) [ 49 , 74 ].…”