IMMUNOCHEMICAL AND IN-SITU HYBRIDIZATION ANALYSES OF RETINOIC ACID RECEPTOR-ALPHA, RECEPTOR-BETA, AND RECEPTOR-GAMMA IN MURINE HARDERIAN AND SUBMANDIBULAR GLANDS

Retinoic acid (RA), through its cognate receptors (retinoic acid recep tors,RARs), plays an important role in the ontogenesis and maintenance of the normal function of murine Harderian and submandibular glands. In the present study, autoradiography was used to study RA binding to these glands. Both glands showed high radioactive labelling after [C-1 4]-RA administration in normal and partially vitamin A-deficient (VAD) mice. The peak uptake was at 6 h after [C-14]-RA administration in no rmal mice and at 0.5 h in VAD mice. At 24 h, RA binding remained high in normal mice, while it decreased significantly in VAD mice. In weste rn blots with an antibody recognizing all forms of RARs, a band of mol ecular weight 51 kDa was seen in homogenates of both glands. Immunohis tochemically, RAR staining was found in the nuclei of the glandular ce lls. The Harderian gland exhibited more intense staining than the subm andibular gland. In the latter, the most intense staining was seen in the acinar cells, followed by the intercalated duct cells. The granula r convoluted tubule showed weak immunostaining and the striated duct w as negative. In the Harderian gland, RAR immunostaining was observed i n both type I and II cells, but only part of them stained with RAR ant ibody. The expression of RAR alpha, beta, and gamma transcripts was st udied by in situ hybridization using specific oligonucleotide probes. The cell-specific expression of RAR alpha mRNA in the submandibular gl and corresponded to the RAR proteins detected by immunohistochemistry, while the RAR beta transcript was mainly seen in the striated duct. T he transcripts of RAR alpha and beta were evenly distributed in type I and II glandular cells of the Harderian gland. RAR gamma labelling wa s below detectable levels in both glands. This result suggests that RA and RARs regulate the functions of Harderian and submandibular glands in a cell-specific manner.