Nobuyuki Masumoto

Magnesium sulfate inhibits oxytocin-induced calcium mobilization in human puerperal myometrial cells: Possible involvement of intracellular free magnesium concentration

OBJECTIVE Our purpose was to elucidate the mechanisms of the tocolytic action of Mg2+ on the human myometrium. STUDY DESIGN The effects of extracellular Mg2+ on oxytocin-induced increases in the intracellular free Ca2+ concentration in human puerperal myometrial cells were examined by means of indo-1-AM. The changes in the intracellular free Mg2+ concentration under various conditions were also measured with Mg(2+)-fura-2-AM. RESULTS The increase in intracellular free Ca2+ concentration induced by oxytocin was reduced to 26% of that in the normal solution 20 minutes after replacement of the normal solution with an extracellular Mg2+ solution, 10 mmol/L. When extracellular Ca2+ was removed, the increase in intracellular free Ca2+ concentration was not suppressed even 20 minutes after the replacement. A solution of extracellular Mg2+ concentration, 10 mmol/L, raised the intracellular free Mg2+ concentration gradually, by approximately 150% in 20 minutes, concomitant with the suppression of the response to oxytocin in the intracellular free Ca2+ concentration. CONCLUSION High intracellular free Mg2+ concentration, caused by high extracellular Mg2+, is essential for suppression of oxytocin-induced Ca2+ influx across the cell membrane; this presumably results in inhibition of uterine contractions.

SNAP-25 is essential for cortical granule exocytosis in mouse eggs

Synaptosome-associated protein of 25 kDa (SNAP-25) has been shown to play an important role in Ca2+-dependent exocytosis in neurons and endocrine cells. During fertilization, sperm-egg fusion induces cytosolic Ca2+ mobilization and subsequently Ca2+-dependent cortical granule (CG) exocytosis in eggs. However, it is not yet clear whether SNAP-25 is involved in this process. In this study, we determined the expression and function of SNAP-25 in mouse eggs. mRNA and SNAP-25 were detected in metaphase II (MII) mouse eggs by RT-PCR and immunoblot analysis, respectively. Next, to determine the function of SNAP-25, we evaluated the change in CG exocytosis with a membrane dye, tetramethylammonium-1,6-diphenyl-1,3,5-hexatriene, after microinjection of a botulinum neurotoxin A (BoNT/A), which selectively cleaves SNAP-25 in MII eggs. Sperm-induced CG exocytosis was significantly inhibited in the BoNT/A-treated eggs. The inhibition was attenuated by coinjection of SNAP-25. These results suggest that SNAP-25 may be involved in Ca2+-dependent CG exocytosis during fertilization in mouse eggs.

Expression of Epidermal Growth Factor and Transforming Growth Factor-Alpha in Fallopian Tube Epithelium and Their Role in Embryogenesis

We studied the expression of epidermal growth factor (EGF) and transforming growth factor (TGF)-alpha in human fallopian tube epithelium at various menstrual stages. Immunohistochemical staining using anti-EGF and anti-TGF-alpha antibodies showed a specific staining in ampullary tube epithelium at late follicular and luteal stages but the staining was very weak at the early follicular stage. Quantitative reverse transcription and polymerase chain reaction (RT-PCR) using beta-actin mRNA as an internal standard revealed the menstrual-stage-specific expression of EGF and TGF-alpha gene transcripts: amounts of EGF and TGF-alpha mRNA relative to those of beta-actin were significantly higher at late follicular and luteal stages than at the early follicular stage. To clarify the biological role of these growth factors, mouse 2-cell embryos were cocultured with human fallopian tube epithelial cells with or without blocking the action of these growth factors. Cocultures significantly promoted blastocyst formation, but this promotive effect of the tubal epithelial cells was completely abolished by the addition of anti-EGF and/or anti-TGF-alpha monoclonal neutralizing antibodies to the coculture system. These results demonstrated that EGF and TGF-alpha were synthesized and expressed in fallopian tube epithelium at specific menstrual stages, and may be involved in early embryonic development.

Simultaneous measurements of exocytosis and intracellular calcium concentration with fluorescent indicators in single pituitary gonadotropes

Previously, we established a method for the estimation of exocytosis in single gonadotropes using an impermeable fluorescent membrane probe, TMA-DPH. In this study, we have developed a method for the simultaneous measurement of exocytosis and intracellular free Ca2+ concentration ([Ca2+]i) by double-labeling with TMA-DPH and the intracellular Ca2+ probe, Fura-2/AM, using a fluorescence microscope with a 3-wavelength excitation and 2-wavelength emission system. We, therefore, clarified the relationship between spontaneous [Ca2+]i oscillation or gonadotropin releasing hormone (GnRH)-induced intracellular Ca2+ mobilization and exocytosis in gonadotropes. Under resting conditions, some gonadotropes showed various types of spontaneous [Ca2+]i oscillations, while others did not, but all showed basal exocytosis. Each [Ca2+]i peak oscillation did not cause Ca(2+)-regulated exocytosis, and even complete blockage of the [Ca2+]i increase by the intracellular Ca2+ chelator BAPTA/AM had no effect on basal exocytosis. Both GnRH-induced intracellular Ca2+ mobilization and regulated exocytosis showed a similar pattern of peaks and plateaus. Blockage of the [Ca2+]i increase by BAPTA/AM almost completely inhibited the GnRH-stimulated exocytosis. These results show that spontaneous [Ca2+]i oscillations under resting conditions are not linked to regulated or basal exocytosis, and that intracellular Ca2+ mobilization is essential for GnRH-stimulated exocytosis.

EXPRESSION OF RAB3A IN THE CORTICAL REGION IN MOUSE METAPHASE II EGGS

Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in regulated exocytosis at presynapses. At fertilization, sperm-egg fusion induces cytosolic calcium mobilization and cortical granule (CG) exocytosis in the egg. However, it is not yet clear whether Rab3A is involved in this process. We previously reported that Rabphilin-3A functions in calcium-dependent CG exocytosis. Rabphilin-3A is known to bind with Rab3A, Rab3B, and Rab3C. In this study, we clarified which member of the Rab3 was expressed in mouse metaphase II eggs. Messenger RNA encoding Rab3A but not Rab3B or Rab3C, was detected in unfertilized metaphase II eggs by RT-PCR. Rab3A protein was also detected in unfertilized metaphase II eggs by immunoblot analysis. Next the expression and the localization of Rab3A in eggs at various stages of development were determined by immunofluorescence analysis using confocal laser scanning microscopy. Rab3A protein was specifically distributed in the cortical region in eggs from before fertilization to the two-cell stage. However, it was not detected at the three- or four-cell stage 40 hr after fertilization. These results suggest that Rab3A may function with Rabphilin-3A in CG exocytosis.

Activin a increases cytosolic free calcium concentration in rat pituitary somatotropes

The effect of activin A on the cytosolic free calcium concentration ([Ca2+]i) in normal rat pituitary cells was examined using a calcium sensitive fluorescent dye, indo 1 AM, and a digital imaging fluorescent microscope system. The cells showing an increase in [Ca2+]i in response to activin A were then characterized by comparison with cells responding to growth hormone releasing hormone (GRH), thyrotropin releasing hormone (TRH), corticotropin releasing hormone (CRH), and gonadotropin releasing hormone (GnRH) in monolayer cultures of normal rat pituitary cells. Activin A increased [Ca2+]i in some cells in a mixed population of normal rat pituitary cells. The cells that responded to activin A also responded to GRH. Most of these cells were not affected by other tropic hormones (CRH, TRH, and GnRH), but a few cells responded to both GRH and TRH. None of the activin A-responding cells responded to CRH or GnRH, and none of the CRH- or GnRH-responding cells responded to activin A. In a preparation of somatotropes purified 80-90% by fluorescence-activated cell sorting, activin A increased [Ca2+]i in 30% of the cells that shows a [Ca2+]i-response to GRH. These findings suggest direct involvement of somatotropes in activin A-induced biological events in the rat pituitary gland.

Effects of thyrotropin-releasing hormone and phorbol ester on dopamine release from dispersed rat tuberoinfundibular dopaminergic neurons.

We have investigated the intracellular mechanisms underlying thyrotropin-releasing hormone (TRH)-mediated [3H]dopamine ([3H]DA) release from dispersed rat tuberoinfundibular dopaminergic (TIDA) neurons. The specific binding of [3H]Me-TRH to these cells is characterized by a single, high-affinity binding site (Kd = 1.2 nM) with a Bmax value of 178 fmol/mg protein. Thyrotropin-releasing hormone markedly increased [3H]DA release and intracellular free calcium concentration ([Ca2+]i) in TIDA neurons, and its effect was abolished by treatment with EGTA (5 mM) or chlordiazepoxide, a specific TRH receptor antagonist (10 microM). Furthermore, to examine the involvement of protein kinase C on [3H]DA release, we investigated the effect of phorbol myristate acetate (PMA), which is known to activate protein kinase C directly. Phorbol myristate acetate induced a significant increase in [3H]DA release in a concentration-dependent manner. Treatment with TRH (1 microM) plus PMA (100 nM) resulted in an additive increase in [3H]DA release. Thyrotropin-releasing hormone (1 microM) still increased [3H]DA release even after preincubation with PMA (500 nM) for 24 h, but PMA (100 nM) did not under the same conditions. These results suggest that TRH may induce DA release in dispersed rat TIDA cells by increasing calcium influx and activating the protein kinase C system.

Roles of prostaglandins and intracellular free calcium mobilisation in epidermal growth factor-induced proliferation of human amnion cells

Objective To investigate the mechanisms which regulate the growth of human amnion cells.