The paracrine effects of cytokines, myokines, and adipokines secreted by immune cells, muscle cells, and adipocytes, respectively, have attracted considerable recent research attention (44C46). 11-hydroxylase (cytochrome P450 111; CYP111), which mediated the production of corticosterone from deoxycorticosterone. However, CYP family 11 subfamily A member 1 (CYP11A1)-mediated production of pregnenolone from cholesterol was not H3B-6527 recognized in H3B-6527 the salivary glands by immunoblotting using a specific antibody. These results indicate that corticosterone and testosterone are produced from pregnenolone in rat salivary glands. The initial substrate in salivary steroidogenesis and the functions of salivary corticosterone and testosterone are discussed. = 0.893) with plasma CORT levels (28, 29). As collection is definitely non-invasive and easy, it is clinically useful to use saliva as a substitute for blood in determining CORT and cortisol levels for diagnostic purposes (30). Measurement of CORT and cortisol levels in saliva is considered superior for diagnostic purposes because it avoids potential artifacts associated with blood draw stress. However, the concentration of CORT in the blood does not totally correlate with the concentration in saliva (31). The significance of salivary CORT is definitely therefore unfamiliar. As an initial step in elucidating the significance of salivary CORT, consequently, the present study provides the first statement of enzymatic activities associated with salivary steroidogenesis in rats. Materials and Methods Chemicals and Reagents The following were purchased from Sigma-Aldrich (St. Louis, MO, USA): precursors of CORT, including PGN, PGS, DCORT; precursor of testosterone (TS), androstenedione (ASD); and conjugated metabolites, pregnenolone-sulfate (PGN-S). Rabbit polyclonal to Caspase 1 Cholesterol was purchased from Wako Pure Chemical Industries (Osaka, Japan). LC-MSCgrade acetonitrile and formic acid were purchased from Supelco (Bellefonte, PA, USA). Ethics Statement This study was carried H3B-6527 out in strict accordance with the recommendations in the Guideline for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The protocol was authorized by the Committee within the Ethics of Animal Experiments of the Rakuno Gakuen University or college (permit quantity: VH17A4). All surgeries were performed under isoflurane anesthesia, and all efforts were made to minimize animal suffering. Preparation of Rat Organs Male (300C400 g) or pregnant female (240C300 g at gestation day time 12C13) Sprague-Dawley rats (8 to 15 weeks-old) were purchased from Sankyo Lab Co. (Tokyo, Japan). The rats were fed, housed, and allowed to adapt to their environment for 1 week before the experiments. The salivary glands (SGs), adrenal glands (Adrs), and testes were collected from your male animals, and placentas were collected from your pregnant females, after euthanasia by exsanguination under isoflurane anesthesia. After dissection, the organs were excised post-mortem and then weighed. SGs utilized for enzyme reaction assays included the sublingual glands (SLGs), submandibular glands (SMGs), and parotid glands (PGs). For the European blot analyses, the PGs were isolated and used separately as PG and SLG+SMG components. A total of 11 male rats were utilized for enzyme reaction assays (6 animals) and Western blot analyses (5 animals), and a total 4 pregnant woman rats were utilized for enzyme reaction assays or Western blot analyses. Antibodies For Western blot analyses, the following antibodies were used: rabbit polyclonal anti-GAPDH (sc-25778, Santa Cruz), anti-CYP11A1 (ab175408, Abcam), anti-StAR (sc-25806, Santa H3B-6527 Cruz), anti-steroid sulfatase (bs-3857R, Bioss), and anti-CYP111 (provided by Dr. Mukai) as main antibodies; and goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (#1706515, BIO-RAD) was used as the secondary antibody. Anti-CYP111 was raised in rabbits by immunization using the peptide related to amino acid residues 272-283 (KNVYRELAEGRQC). For affinity preparation of the antibody, the sulfhydryl group of the carboxy terminal cysteine residue H3B-6527 was conjugated with carrier protein (32). Dedication of Steroid Levels in the SGs Samples were prepared for MS analysis of steroids.