Gonadotrophin-releasing hormone (GnRH) induces the release of gonadotrophins via an increase in cytosolic Ca 2+ concentration ([Ca 2+ ]). Rab3B, a member of the small GTP-binding protein Rab family, is known to be involved in Ca 2+ -regulated exocytosis in pituitary cells. However, it is not known whether Rab3B functions in the physiological process regulated by GnRH in gonadotrophs. In this study using antisense oligonucleotide against Rab3B (AS-Rab3B) we determined that Rab3B is involved in GnRH-induced gonadotrophin release. Rab3B immunopositive cells were reduced in 24% of pituitary cells by AS-Rab3B. This treatment did not affect the population of gonadotrophs or the intracellular contents of gonadotrophins. However, AS-Rab3B significantly inhibited the total amount of basal and GnRH-induced gonadotrophin released from pituitary cells. These results show that Rab3B is involved in basal and GnRH-induced gonadotrophins release but not the storage of gonadotrophins. Next, the changes in [Ca 2+ ] and exocytosis in gonadotrophs treated with AS-Rab3B were compared among Rab3B-positive and -negative cells. The change in [Ca 2+ ] was not different in the two groups, but exocytosis was significantly inhibited in Rab3B-negative cells. These results suggest that Rab3B is essential for GnRH-regulated exocytosis downstream of cytosolic Ca 2+ in gonadotrophs.

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Abstract. The present study was designed to characterize bihormonal cells in rat pituitary cells which secrete PRL and gonadotropins.This was done by using sandwich cell immunoblot assay (CIBA) and reverse hemolytic plaque assay (RHPA) in combination with immunocytochemistry (ICC) and by measuring the intracellular free calcium concentration ([Ca2+];). The result of the experiment with sandwich CIBA revealed that the populations of LH-and FSH-secreting cells in the PRL-secreting cells were 6.23% and 5.91%, respectively, and the populations of the PRL secreting cells in the LH-and FSHsecreting cells were 18.4% and 15.5%, respectively. Additional studies by the combined techniques of RHPA with ICC revealed that the populations of LH-and FSH-containing cells in the PRL secreting cells were 4.43% and 2.40%, respectively, which were consistent with the results of Sandwich CIBA. Some of the PRL-secreting cells determined by RHPA showed responsiveness to TRH and GnRH in [Ca2+];. These results suggest that bihormonal cells which secrete both PRL and LH, or both PRL and FSH are present in the normal rat pituitaries.