CALMODULIN, CALBINDIN-D28K AND CALRETININ IN RAT AND CHICKEN PINEAL GLANDS - IMMUNOCYTOCHEMICAL AND IMMUNOBLOTTING ANALYSIS

In pineal gland, melatonin is synthesized in pinealocytes. Pharmacolog ical studies using calmodulin antagonists suggested that melatonin syn thesis was regulated through calmodulin. However, immunohistochemical studies showed that calmodulin could only be detected in pineal glial cells, and not in pinealocytes. To further investigate this discrepanc y, we have tried to detect calmodulin not seen by immunohistochemical methods. We have used rat and chicken pineal homogenate supernatants a nd Triton X-100-treated pellets denatured by sodium dodecyl sulfate, s ubjected to electrophoresis and immunoblotting using anti-calmodulin a ntibodies. Two different IgG (#465 and #860) purified from anti-calmod ulin sera were used. In rat pineal homogenate supernatants, calmodulin could be detected by immunoblotting using both antibodies. Some calmo dulin could also be detected in the Triton-treated pellet fractions, b ut no additional cross-reacting bands were detected. However, in both chicken pineal homogenate supernatants and Triton-extracted pellets, i n addition to a calmodulin immunoreactive band, two other proteins wit h approximate molecular masses (M(r)) of 56 kDa and 60 kDa were detect ed using anti-calmodulin #465. For comparison, similar immunoblot expe riments were performed for detection of calbindin-D28K and calretinin, two other calcium binding proteins expressed in different pineal cell populations. Interestingly, Triton extraction of chicken pineal pelle ts revealed additional bands cross-reacting with each antibody. Anti-c albindin-D28K cross-reacted strongly with a M(r) = 68 kDa protein and weakly with a M(r) = 56 kDa protein. Anti-calretinin cross-reacted str ongly with a M(r) = 93 kDa protein and weakly with a M(r) = 56 kDa pro tein.