Our previous studies on annexin 5, a member of the annexin family of proteins, have shown its expression in the anterior pituitary gland, its preferential distribution in gonadotropes, and its increase after ovariectomy. In the present study, we examined (1) whether annexin 5 is synthesized in gonadotropes, (2) whether its expression is under the control of gonadotropin-releasing hormone (GnRH), and (3) the effect of annexin 5 on gonadotropin release. Large cells, also called castration cells, appeared in anterior pituitary tissue 3 weeks after ovariectomy. These cells have been confirmed to be hyperfunctioning gonadotropes and are easily discriminated from other pituitary cells without immunostaining. Using in situ hybridization with a digoxigenin-labeled ribonucleic acid probe, enhanced expression of annexin 5 messenger ribonucleic acid (mRNA) in these gonadotropes was clearly demonstrated. Northern blot analysis showed an increase in the level of annexin 5 mRNA expression 3 weeks after ovariectomy. It was lessened 3 h after the injection of Cetrorelix (GnRH antagonist, 10 µg i.v.). Administration of a GnRH analog [GnRHa; Des-Gly 10 (Pro9) GnRH ethylamide, 0.2 ml of 2.5 µg/ml saline ten times intraperitoneally at 30-min intervals] significantly increased pituitary annexin 5 mRNA. In primary cultures of anterior pituitary cells, recombinant rat annexin 5 stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release in a dose-dependent manner. Concomitant administration of annexin 5 (1 µg/ml) and GnRHa augmented the LH and FSH release induced by GnRHa. After a 1-hour incubation, cycloheximide (10 µg/ml) apparently inhibited the LH response to GnRHa, while annexin 5 (2 µg/ml) moderated this inhibition. Further, the antisense oligodeoxynucleotide to annexin 5 mRNA blunted the LH response to GnRHa. It is thus concluded that annexin 5 is synthesized in the gonadotropes under the effect of GnRH, and it is suggested that annexin 5 synthesis mediates at least partly GnRH receptor signaling to stimulate gonadotropin secretion.

Three metabolites were isolated from the culture broth of an actinomycete strain identified as Streptomycesplatensis SANK601 91 , that induce the production of colony-stimulating factors (CSFs) by stromal cell line KM-102at ED50concentrations from 40 to 200 ng/ml. The compounds induced quantities of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) comparable to those induced by interleukin-1 , a strong CSFinducer. These metabolites were called leustroducsins (A, B and C) and were later found to be structurally related to phoslactomycins. This is the first report of CSFinducing activity by members of the phoslactomycin class. 1503Recent studies have demonstrated the application of colony-stimulating factors (CSFs) in clinical use1}. These substances have been used to recover the peripheral blood leukocytes in leukopenia patients caused by cancer-chemotherapy, radiotherapy and bone marrowtransplantation. WhenCSFs were administered to patients, restorations ofleukocyte counts occurred. However,the in vivo role ofendogenous CSFs is not precisely understood. In particular, little is known about the regulatory mechanism for CSF production or about the relationship between normal blood cell production and endogenous CSFs. It is well knownthat bone marrow stromal cells play an important role in hematopoiesis. Regulation of CSF production by bone marrow stromal cells may be one of the key elements responsible for the control of hematopoiesis in vivo2). Therefore, substances that affect the regulation of CSF production by stromal cells are of potential interest, so we worked to develop screening methods for CSF inducers.In the previous paper3), we described the development of a new screening method for CSF inducers using human bone marrow stromal cell line KM-102. Using this screening method, one strain of actinomycetes, Streptomyces platensis SANK60191, was found to produce novel microbial metabolites that we provisionally namedleustroducsins. Structure determination studies revealed that they are congeners of phoslactomycins. In this paper we report the taxonomy of the producing organism, and also describe the fermentation and biological properties of leustroducsins (LSNs), A, B and C (Fig. 1). The isolation, physicochemical properties and structural elucidation of the metabolites are reported in the accompanying paper4).

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Streptomyces hygroscopicus subsp. aureolacrimosus SANK 60286 produces a new family of milbemycins, named milbemycins an, p12, a13, a14 and a15, together with other milbemycins. Their structures are 3-methyl-2-butenoyloxy and 3-methylbutyroyloxy derivatives at C-4a of milbemycins A3 and A4, or 3-methyl-2-pentenoyloxy derivative at C-4a of milbemycin A3, respectively. Milbemycin a 1 4, 3-methyl-2-butenoyloxy derivative, especially possesses a potent acaricidal activity.

Abstract. Annexin 5, a novel calcium-phospholipid binding protein, is thought to be involved in hormone secretion by the anterior pituitary gland. Gonadotropin releasing hormone stimulates annexin 5 synthesis, which, in turn, enhances gonadotoropin secretion. On the other hand, annexin 5 was shown to inhibit prolactin release in vitro. To understand the nature of the opposing effects of annexin 5 on these two major pituitary hormones, the present study examines the inhibitory effect of annexin 5 on prolactin release in relation to thyrotropin stimulating hormone (TRH) using primary cultures of anterior pituitary cells of adult female rats. While recombinant rat annexin 5 was found to have little effect on basal prolactin release, it significantly inhibited TRH-stimulated prolactin release. Addition of specific anti-annexin 5 serum to the culture increased basal prolactin release in a concentration dependent manner, and no further increase in prolactin release was observed following application of TRH in the presence of anti-annexin 5. The enhanced basal prolactin release induced by anti-annexin 5 was reversed by the simultaneous administration of indomethacin, an inhibitor of cyclooxygenase. These results demonstrate that endogenous pituitary annexin 5 exerts an inhibitory effect on prolactin release and suggest that this is attained by suppression of eicosanoid synthesis in vitro.