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The effect of prostaglandin D2 (PGD2) on release of LH and LH releasing hormone (LHRH) was studied in a sequential double-chamber superfusion system using the medial basal hypothalamus (MBH) and the pituitary gland from female rats at dioestrus. Infusion of PGD2 (5·7 or 57μmol/l) caused a significant (P <0·05) increase in LH release to values 40–60% above the preinjection values from the pituitary gland superfused either alone or in series with the MBH. No release of LHRH in response to PGD2 was observed from the superfused MBH. These data demonstrate that PGD2 causes LH release from the pituitary gland not by inducing release of hypothalamic LHRH but by a direct action on the gland.
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Abstract
These studies were undertaken to characterize the exocytotic changes in purified gonadotropes by three-dimensional imaging using scanning electron microscopy. Rat gonadotropes were purified using a fluorescence-activated cell sorter and an argon laser treatment system. The purified gonadotropes were stimulated with GnRH under various conditions and fixed for scanning electron microscopy. After the GnRH stimulation, many 'hole' structures (diameter 0·1–0·5 μm) were observed on the cell surface, and notably the population of cells with 10 or more holes was clearly increased. The pattern of the time-course of the changes in this population was perfectly consistent with the LH secretory profile of pituitary cells, and their formation of the cells with 10 or more holes was completely inhibited by pretreatment with a GnRH antagonist. Our data suggest that the hole structure represents an exocytotic opening site and that regulated exocytosis in purified gonadotropes can be evaluated by scanning electron microscopy. This method may be widely applicable to other endocrine cells.
Journal of Endocrinology (1995) 144, 193–200
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Abstract
The regulation of mouse placental lactogen (mPL)-I and mPL-II secretion by activin and inhibin and the expression of activin and inhibin subunit mRNAs in the mouse decidua were examined. Activin-A at a concentration of 10 nm/l significantly inhibited mPL-II secretion by placental cells from days 9 and 12 of pregnancy. However, activin-A did not affect mPL-I secretion by cells from days 7 and 9 of pregnancy nor mPL-II secretion by cells from day 7 of pregnancy. By contrast, 10 nm/l inhibin activated mPL-II secretion by cells from day 12 of pregnancy. These effects of activin and inhibin on mPL-II secretion were dose-dependent. Follistatin, which binds to activin and blocks its bioactivity, completely eliminated the inhibitory effect of activin on mPL-II secretion. Incubation of placental cells from day 12 of pregnancy with activin-A resulted in a significant reduction of the mPL-II mRNA level assessed by Northern blot analysis. Northern blot analysis using poly(A)+ RNA extracted from the decidua indicated that mouse decidua, as well as the placenta, express all activin and inhibin subunits and that their gene expressions increased during gestation. The expression of these mRNAs in the decidua was much higher than those in the placenta. These findings suggest that activin and inhibin regulate mPL-II secretion and suggest the presence of an autocrine or paracrine regulation of mPL-II secretion in mouse placenta by activin and inhibin after mid-pregnancy in vivo.
Journal of Endocrinology (1995) 146, 469–474
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Abstract
The synaptic membrane protein synaptosomal-associated protein (SNAP-25) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion in neurons. However, the role of SNAP-25 in pituitary hormone release is not known. In this study, we determined that SNAP-25 is involved in regulated exocytosis in the clonal pituitary cell line GH4C1. SNAP-25 messenger RNA and protein were detected in GH4C1 cells by RT-PCR and immunoblot analysis, respectively. Immunofluorescence analysis indicated that SNAP-25 protein was localized in the plasma membrane. Next, to determine the function of SNAP-25 in GH4C1 cells, specific inhibitors of SNAP-25, botulinum neurotoxin (BoNT) /A or /E, and antisense SNAP-25 oligonucleotide were used. Neither BoNT/A nor BoNT/E affected thyrotropin-releasing hormone (TRH)-induced cytosolic Ca2+ increase, but both inhibited TRH-induced exocytosis. Moreover, they dose-dependently inhibited TRH-induced prolactin release. The introduction of antisense oligonucleotide into the cells also inhibited TRH-induced prolactin release. These results suggest that SNAP-25 is involved in regulated exocytosis in GH4C1 cells.
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Gonadotrophin-releasing hormone (GnRH) induces the release of gonadotrophins via an increase in cytosolic Ca2+ concentration ([Ca2+]). 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 [Ca2+] and exocytosis in gonadotrophs treated with AS-Rab3B were compared among Rab3B-positive and -negative cells. The change in [Ca2+] 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 Ca2+ in gonadotrophs.
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Using digitonin-permeabilized GH3 cells, we investigated both the release of prolactin (PRL) and changes in the cytoskeleton. We determined that permeabilized GH3 cells released PRL in a dose-dependent manner upon addition of micromolar Ca(2+). Phalloidin, a filamentous actin (F-actin) stabilizing agent, inhibited both Ca(2+)-dependent and -independent PRL release, whereas cytochalasin B, a destabilizing agent, had almost no effect on the release. Observation with a confocal laser scanning microscope revealed that F-actin existed mainly in the cortical region in the quiescent state. Increased cytosolic Ca(2+) induced a change in F-actin distribution: F-actin in the cortical region decreased, whereas F-actin inside the cells increased. This change in F-actin distribution was not observed when phalloidin was added. Addition of cytochalasin B induced patchy F-actin spots, but the pattern of the changes of F-actin distribution did not change. The time course of change in F-actin distribution showed that the F-actin network in the cortical region was reduced within 1 min, and Ca(2+)-dependent release of PRL continued for up to 20 min. These results suggest that the F-actin network near the membrane acts as a barrier to exocytosis and that Ca(2+) directly controls the cytoskeletal changes.
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Breast milk has non-nutritional protective effects on recipient infants. It has been speculated that bioactive substances present in human milk have important roles in protecting infants. However, the mechanisms by which such substances protect newborns are unclear. Therefore, we analyzed the growth-promoting activity of human milk and the intracellular signaling mechanism thereof using human fetal small intestinal (FHS 74 Int) cells. Epidermal growth factor (EGF) stimulated the proliferation of these cells. However, this stimulation was less effective than that of aqueous milk (5% vol/vol). The bioactivity of human milk was heat stable but protease sensitive. EGF receptor tyrosine kinase inhibitor did not repress the milk-induced growth-promoting effect on fetal small intestinal cells. Regarding the intracellular signaling pathway, the milk-induced cell proliferation pathway was tyrosine kinase dependent but was neither mitogen-activated protein (MAP) kinase nor phosphatidylinositol-3 (PI-3) kinase dependent. On the other hand, EGF-induced cell proliferation was tyrosine kinase, MAP kinase, and PI-3 kinase dependent. Rapid tyrosine phosphorylation of several intracellular proteins was detected after milk stimulation. Furthermore, the time course of phosphorylation induced by milk was different from that induced by EGF. The sizes of the proteins phosphorylated in response to milk were different from those of the Shc proteins phosphorylated in response to EGF. These results suggest that human milk induces fetal intestinal cell proliferation through a unique tyrosine kinase pathway different from the EGF receptor signaling pathway.
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The proliferation of vascular smooth muscle cells (VSMC) is a crucial pathophysiological process in the development of atherosclerosis. Although estrogen is known to inhibit the proliferation of VSMC, the mechanism responsible for this effect remains to be elucidated. In addition, the effect of raloxifene on VSMC remains unknown. We have shown here that 17beta-estradiol (E(2)) and raloxifene significantly inhibited the platelet-derived growth factor (PDGF)-stimulated proliferation of cultured human VSMC. Flow cytometry demonstrated that PDGF-stimulated S-phase progression of the cell cycle in VSMC was also suppressed by E(2) or raloxifene. We found that PDGF-induced phosphorylation of retinoblastoma protein (pRb), whose hyperphosphorylation is a hallmark of the G1-S transition in the cell cycle, was significantly inhibited by E(2) and raloxifene. These effects were associated with a decrease in cyclin D1 expression, without a change in cyclin-dependent kinase 4 or cyclin-dependent kinase inhibitor, p27(kip1) expression. ICI 182,780 abolished the inhibitory effects of E(2) and raloxifene on PDGF-induced pRb phosphorylation. Next, we examined which estrogen receptor (ER) is necessary for these effects of E(2) and raloxifene. Since VSMC express both ERalpha and ERbeta, A10, a rat aortic smooth muscle cell line that expresses ERbeta but not ERalpha, was used. The dose-dependent stimulation of A10 cell proliferation by PDGF was not inhibited by E(2) or raloxifene in contrast to the results obtained in VSMC. Moreover, E(2) and raloxifene significantly inhibited the PDGF-induced cyclin D1 promoter activity in A10 cells transfected with cDNA for ERalpha but not in the parental cells. These results suggested that E(2) and raloxifene exert an antiproliferative effect in VSMC treated with PDGF, at least in part through inhibition of pRb phosphorylation, and that the inhibitory effects of E(2) and raloxifene may be mainly mediated by ERalpha.
