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On the basis of our previous observation that estrogen stimulates calcitonin secretion by the ultimobranchial gland in the stingray, Dasyatis akajei, experiments were conducted to examine the presence of estrogen receptor (ER) and its mRNA in the gland, employing exchange assay and Northern blot analysis, respectively. The optimal incubation conditions of the exchange assay for cytosolic ER were found to be 25 degrees for 2 hr. Scatchard analysis of cytosolic ER revealed two components with dissociation constants (Kd) of 0.090 +/- 0.015 and 17.780 +/- 1.910 nM and with a maximum number of binding sites (Bmax) of 62.2 +/- 7.9 and 2290 +/- 180 fmol/mg protein, respectively. Furthermore, Sephadex G-100 filtration of the cytosol and immunoblot analysis using antiserum against a recombinant rat ER confirmed the presence of two specific binding components for estrogen (41 and 57 kDa). Total RNA extracted from the ultimobranchial gland was subjected to Northern blot analysis using a rat ER cDNA as a probe. Two positive signals were detected at 2.5 and 2.0 kb. Thus, the presence of ER was confirmed for the first time in nonmammalian calcitonin-secreting cells.
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