Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.

Involvement of Sp-1 in the regulation of the Id-1 gene during trophoblast cell differentiation.

Id-1, a member of the helix-loop-helix transcription factor family, inhibits the differentiation of Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells. Id-1 is expressed at a high level in undifferentiated trophoblast stem cells and then down-regulated during early differentiation, and is thought to be a key regulator in the trophoblast giant-cell differentiation pathway. In this study, we analyzed the signaling mechanism regulating the high expression levels of Id-1 in undifferentiated Rcho-1 cells. Promoter deletion analysis revealed that a 31-bp sequence (Box-2 region), located between -200 and -169bp in the Id-1 promoter is necessary for the promoter activity. Electrophoretic mobility shift assays and DNA affinity precipitation assays showed that Box-2-binding activity was decreased during differentiation and that Sp-1 protein bound to this sequence. The protein level of Sp-1 was decreased during the differentiation. These results suggest that the Sp-1 protein level may regulate the Box-2-binding activity and the trophoblast giant-cell differentiation.

STAT3-mediated constitutive expression of SOCS3 in an undifferentiated rat trophoblast-like cell line.

In the trophoblast, constitutive expression of SOCS3 is important for the negative regulation of trophoblast giant cell differentiation. In this study, we analyzed the signaling pathway regulating the constitutive SOCS3 expression in undifferentiated Rcho-1 cells, which were derived from rat choriocarcinoma and consist of trophoblast stem cells that are capable of differentiating to trophoblast giant cells in vitro. PD98059, an MEK inhibitor, repressed the SOCS3 expression but AG490, a JAK2 inhibitor, did not. Promoter deletion analysis revealed that the STAT response element (SRE) in the SOCS3 promoter is necessary for the promoter activity. Overexpression of STAT3 increased the SOCS3 promoter activity, whereas expression of dominant-negative STAT3 reduced it. Constitutive STAT3 tyrosine phosphorylation that was not inhibited by either AG490 or PD98059 was demonstrated. Electrophoretic mobility shift assays showed the existence of a protein that bound to SRE and was supershifted with STAT3 antibody. This binding reaction was inhibited by neither AG490 nor PD98059. These findings imply that the ERK/MAPK pathway and STAT3 are involved in the constitutive activation of SOCS3 in undifferentiated Rcho-1 cells. Moreover, they indicate that the constitutive STAT3 tyrosine phosphorylation and the DNA binding activity of STAT3 do not depend on the ERK/MAPK or JAK kinase pathway. These results suggest that a trophoblast-specific STAT3 activation pathway is important for the regulation of giant cell differentiation.

Role of mitogen-activated protein kinase/extracellular signal-regulated kinase cascade in gonadotropin-releasing hormone-induced growth inhibition of a human ovarian cancer cell line.

Although gonadotropin-releasing hormone agonists (GnRHa) have been used in the therapy of the endocrine-dependent cancers, their biological mechanism remained obscure. We have studied the roles of mitogen-activated protein kinase family in the antiproliferative effect of GnRHa on the Caov-3 human ovarian cancer cell line. Reverse transcription-PCR assays confirmed mRNA for GnRH receptor in Caov-3 cells. In the presence of 1 microM GnRHa, the proliferation of cells was significantly reduced to 76% of controls after 24 h, and the effect was sustained up to 4 days. Although GnRHa had no effect on the activation of the Jun N-terminal kinase (JNK), treatment of Caov-3 cells with GnRHa activated extracellular signal-regulated protein kinase (ERK), and its effect was more than that induced by GnRH. Activation of ERK by GnRHa occurred within 5 min, with the maximum occurring at 3 h and sustained until 24 h. GnRHa also activated ERK kinase (mitogen-activated protein/ERK kinase) and resulted in an increase in phosphorylation of son of sevenless (Sos), and Shc. Furthermore, we examined the mechanism by which GnRHa induced ERK activation. Both pertussis toxin (10 ng/ml), which inactivates Gi/Go proteins, and expression of a peptide derived from the carboxyl terminus of the beta-adrenergic receptor kinase I, which specifically blocks signaling mediated by the betagamma subunits of G proteins, blocked the GnRHa-induced ERK activation. Phorbol 12-myristate 13-acetate (PMA) also induced the ERK activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of ERK by GnRHa. Elimination of extracellular Ca2+ by EGTA also did not abolish the activation of ERK by GnRHa. To examine the role of ERK cascade in the antiproliferative effect of GnRHa, PD98059, an inhibitor of mitogen-activated protein/ERK kinase, was used. This inhibitor canceled the antiproliferative effect of GnRHa and apparently reversed the GnRH-induced dephosphorylation of the retinoblastoma protein, the hyperphosphorylation of which is a hallmark of G1-S transition in the cell cycle. These results provide evidence that GnRHa stimulation of ERK activity may be mediated by Gbetagamma protein, not by PMA-sensitive protein kinase C nor extracellular Ca2+ in the Caov-3 human ovarian cancer cell line, suggesting that this cascade may play an important role in the antiproliferative effect of GnRHa.

Inhibition of BAD phosphorylation either at serine 112 via extracellular signal-regulated protein kinase cascade or at serine 136 via Akt cascade sensitizes human ovarian cancer cells to cisplatin.

We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-protein kinase B/Akt-BAD cascade in both cisplatin-resistant Caov-3 and -sensitive A2780 human ovarian cancer cell lines. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum (transplatin) isomer stimulated the activation of Akt, and the PI-3K inhibitor wortmannin blocked the cisplatin-induced activation of Akt. Treatment of both Caov-3 and A2780 cells with cisplatin but not with the trans-diaminodichloroplatinum isomer also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. Whereas the phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin, its phosphorylation at Ser-112 was blocked by a MAP/ERK kinase inhibitor, PD98059. Exogenous expression of a dominant-negative Akt in both Caov-3 and A2780 cells decreased the cell viability after treatment with cisplatin. In contrast, no sensitization to cisplatin was observed in cells expressing wild-type Akt. We further examined the role of BAD in the viability after cisplatin treatment using BAD mutants. Exogenous expression of each of the singly substituted BADS112A or BADS136A in both Caov-3 and A2780 cells decreased the viability after treatment with cisplatin to a degree intermediate between that caused by exogenous expression of wild-type BAD and doubly substituted BAD2SA. Cisplatin did not stimulate the phosphorylation of BAD Ser-136, but did stimulate the phosphorylation of BAD Ser-112 in cells expressing a dominant-negative Akt, suggesting that BAD Ser-136 but not Ser-112 was phosphorylated by Akt. Our findings suggest that cisplatin-induced DNA damage causes the phosphorylation of both BAD Ser-112 via an extracellular signal-regulated protein kinase (ERK) cascade and BAD Ser-136 via a PI-3K-protein kinase B/Akt cascade and that inhibition of either of these cascades sensitizes ovarian cancer cells to cisplatin.

Permeability increase in black lipid membrane induced by compound 48/80.

When compound 48/80, a potent histamine liberator, was added in the aqueous phase facing the black lipid membrane, the conductivity of the membrane was remarkably increased. Although valinomycin displayed a distinct selectivity for K+ movement, such selection for ionic permeability was not observed in the case of compound 48/80.

Analysis of the mechanism of histamine release induced by substance P.

Substance P causes release of histamine from rat peritoneal mast cells; the structure-activity relationship shows that N-terminal residue is essential and the hydrophobic region of C-terminal plays an important role. Electrical conductivity of black lipid membrane containing phosphatidic acid was augmented by substance P. In this case, N-terminal residues and C-terminal hydrophobicity were also unavoidable. The partitioning of substance P into the organic phase increased in the presence of phosphatidic acid. The CD spectrum of substance P was changed from the unordered form to beta-form by coexistence of phosphatidic acid/PC liposomes in the medium. The addition of calcium or magnesium in the test solution is effective to prevent either of these phenomena. These findings indicate that substance P probably binds to negatively charged sites of membrane lipids, and subsequent penetration of C-terminal into the hydrophobic core of lipid bilayer may induce an increase of membrane permeability and the following histamine release.

Potential mechanisms of resistance to TRAIL/Apo2L-induced apoptosis in human promyelocytic leukemia HL-60 cells during granulocytic differentiation.

Human promyelocytic leukemia HL-60 cells are well known to differentiate into granulocytes or monocytes in the presence of some agents such as DMSO or PMA, respectively. Differentiated HL-60 cells become resistant to some apoptotic stimuli including anticancer drugs or irradiation though undifferentiated cells significantly respond to these stimuli. TRAIL (TNF-related apoptosis-inducing ligand) which is also known as Apo2 ligand (Apo2L), a new member of TNF family, can induce apoptosis in some tumor cells but not in many normal cells. We show here that apoptosis is well induced in HL-60 cells by TRAIL, but susceptibility to TRAIL is reduced during granulocytic differentiation by DMSO. We also suggest some possible mechanisms by which granulocytic differentiated cells become resistant to TRAIL-induced apoptosis. First, in granulocytic differentiated cells, expression of antagonistic decoy receptors for TRAIL (TRAIL-R3/TRID/DcR1/LIT and TRAIL-R4/TRUNDD/DcR2) were enhanced. In addition, expression of Toso, a cell surface apoptosis regulator, seemed to block activation of caspase-8 by TRAIL via enhanced expression of FLIPL in granulocytic differentiated cells. These findings suggest that differentiated cells are resistant using plural mechanisms against various apoptosis-inducing stimuli rather than undifferentiated cells.

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.

Tumor necrosis factor-alpha stimulates prolactin release from anterior pituitary cells: a possible involvement of intracellular calcium mobilization.

We investigated the effects of tumor necrosis factor-alpha (TNF alpha), a cytokine produced as one aspect of inflammatory reactions, on the intracellular free calcium concentration in single pituitary cells using a calcium-sensitive fluorescent dye, indo-1, and a digital imaging fluorescence microscopic system. TNF alpha induced an increase in intracellular free calcium immediately after administration, reaching a peak after 30 sec and then returning to nearly the basal level after 120 sec in the pituitary cells. The cells responding to TNF alpha constitute about 15% of the pituitary cell population, and these cells never responded to the hypothalamic releasing hormones TRH, GnRH, GH-releasing hormone, or CRH. To define the role of calcium in TNF alpha-induced PRL release, dispersed pituitary cells were exposed to agents that modify TNF alpha-induced calcium mobilization. Such calcium channel blockers as cobalt and verapamil decreased basal and TNF alpha-induced PRL release. A low calcium medium also decreased TNF alpha-induced PRL release. These data suggest that intracellular calcium mobilization may be involved in the process of TNF alpha-induced PRL release.

Estrogen induces the release of luteinizing hormone-releasing hormone in normal cyclic women.

The plasma concentrations of immunoreactive LRH, LH, and FSH were determined by RIA every 6 h until 72 h after iv administration of conjugated estrogens during the midfollicular phase. The percentage change in LH from the preinjection level showed a biphasic pattern after the injection of conjugated estrogens, i.e. significant suppression (-70%) from 6-42 h after the injection, followed by a rebound increase with a peak (+150%) at 56 h. Plasma FSH after the injection also showed a biphasic pattern. The plasma immunoreactive LRH levels were unchanged until 32 h after the injection, but then increased significantly (P less than 0.02) to 160% of the preinjection level at 42 h and then decreased rapidly. These data indicate that 1) estrogen administration results in increases in plasma immunoreactive LRH and LH, and the peak of plasma LRH precedes that of gonadotropin; and 2) the negative feedback effect of estrogen on gonadotropin secretion may not be mediated through LRH.

Expression of messenger ribonucleic acid for epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and EGF receptor in human amnion cells: possible role of TGF alpha in prostaglandin E2 synthesis and cell proliferation.

The amnion plays important structural and functional roles in the maintenance of pregnancy and the initiation of parturition. Recently, we reported that epidermal growth factor (EGF) activates prostaglandin (PG) production and cell growth in cultured amnion cells. In this study, we showed the expression of EGF, transforming growth factor-alpha (TGF alpha), and EGF receptor protein and messenger ribonucleic acid in amnion cells, using an immunofluorescence technique and the reverse transcription-polymerase chain reaction. Next, we studied the effect of TGF alpha on intracellular Ca2+ mobilization and PGE2 production in amnion cells. TGF alpha induced an increase in the intracellular Ca2+ concentration in amnion cells, and this increase was significantly reduced when the cells were incubated with cobalt chloride (a Ca2+ channel blocker; 2.5 mmol/L) or EGTA (a Ca2+ chelator; 5 mmol/L). TGF alpha enhanced PGE2 production, and this increase was significantly inhibited when the cells were incubated with indomethacin (a cyclooxygenase inhibitor; 10 mumol/L), cobalt chloride (2.5 mmol/L), or EGTA (5 mmol/L). We also investigated the effect of TGF alpha on the growth of cultured human amnion cells by using flow cytometric analysis of the DNA content. TGF alpha induced DNA synthesis by human amnion cells, and indomethacin inhibited the TGF alpha-induced DNA synthesis. These results suggest that 1) EGF/TGF alpha are expressed and produced in amnion cells; 2) these endogenous factors may regulate the proliferation of amnion cells in an autocrine or paracrine manner; and 3) these growth factors may exert their effects via intracellular Ca2+ mobilization and PGE2 production.

Compound 48/80-induced permeability change in liposomal membrane.

The action of compound 48/80 (a mixture of condensation products of p-methoxy-N-methylphenethylamine with formaldehyde) on liposomal membranes was studied by means of K+-loaded liposomes and a K+ ion-selective electrode. Prompt efflux of K+ was detected when 48/80 was added to the negatively charged liposome suspension, while the monomer of 48/80, p-methoxy-N-methylphenethylamine, did not release K+ from the same liposomes. The mechanism for the action of 48/80 is discussed in comparison with that of a polymyxin, well known as an antibiotic acting on bacterial membranes.

Phosphorylation of smg p21B in rat peritoneal mast cells in association with histamine release inhibition by dibutyryl-cAMP.

IP3 formation and histamine release from rat peritoneal mast cells stimulated by compound 48/80 were dose-dependently inhibited by Bt2cAMP. These inhibitions were restored to the control level in the presence of H-8, a protein kinase A inhibitor. The 22 kDa protein in mast cells was revealed as a markedly phosphorylated protein by incubating with Bt2cAMP, and this phosphorylation was also diminished by H-8. The 22 kDa phosphoprotein of rat mast cells comigrated with phosphorylated smg p21B, purified from human platelets and phosphorylated by protein kinase A in cell-free system, in both one- and two-dimensional PAGE analysis. Moreover, 22 kDa protein in mast cells was identified as smg p21B by immunoblot analysis using an antibody against smg p21B. From the present study, it became clear that smg p21B is phosphorylated by means of protein kinase A system in rat peritoneal mast cells, and it was assumed that phosphorylated smg p21B plays some important role in the suppression of IP3 formation and histamine release from rat peritoneal mast cells.

Presence of non-selective type of endothelin receptor on vascular endothelium and its linkage to vasodilation.

We studied the role of non-selective type (ETB) of endothelin (ET) receptor in the vasculature, using a ligand specific to the ETB receptor, [Glu9]-sarafotoxin S6b ([Glu9]SRTb). Endothelium-containing rat thoracic aorta possessed specific binding sites for 125I-[Glu9]SRTb, which were almost eliminated by removal of the endothelium, while ET-3-specific binding sites were not detected in the endothelium-intact rat aorta. Only ETB receptor was detected in the membranes from the endothelium of porcine thoracic aorta. [Glu9]SRTb exerted only vasodilation in rat aortic ring. These findings indicate that ETB receptors are located on vascular endothelium and linked to vasodilation.

Structure-activity relationship in vasoconstrictor effects of sarafotoxins and endothelin-1.

Sarafotoxins (SRTa, SRTb and SRTc) as well as endothelin-1 (ET-1) produced vasoconstrictions in rat thoracic aorta, rat isolated perfused mesentery and pithed rat in various of extents. The potency was ET-1 greater than SRTb greater than SRTa greater than SRTc at lower doses, but SRTb greater than ET-1 greater than SRTa greater than SRTc at higher doses. [Nitrophenylsulfenylated Trp21]SRTb and SRTb(1-19) caused no vasoconstriction. Either the reduction and carboxymethylation of Cys residues, the destruction of the intramolecular loop or the production of the non-natural disulfide bond, eliminated the constrictor activity. These results indicate that Trp21 and the intramolecular loop structure are essential, and Lys9 and Tyr13 may play some important roles for the vasoconstrictor activity of these peptides.

Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins.

By means of reverse-phase HPLC, 2 different proteins were obtained from apparently purified pig eosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values, although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre-proGMBP2 with 3 domains; signal peptide, acidic pro-portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein; but no such similarity existed in the pro-portion. Although the molecular weights determined by SDS-PAGE of guinea pig and human MBPs were 11,000 and 9,300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the basic domains of mature proteins, were well conserved between guinea pig and human MBPs.

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.

A new method of eliciting delayed hypersensitivity in the mouse abdominal cavity.

A new method of determining delayed hypersensitivity quantitatively was investigated in mice. Mice were sensitized with 150 micrograms of ferritin and, 3 weeks later, antigen challenge was performed by implanting a sponge containing antigen in the abdominal cavity. Cells accumulated in the sponge markedly increased in number for 24-72 h after the challenge; mononuclear cells predominated by 48 h. When sensitized lymphocytes were transferred passively to a normal recipient, marked cell accumulation in the sponge was found 48 h after the challenge. Immunological specificity was confirmed in animals sensitized to antigen and receiving passive transfer of sensitized cells. Strain differences in this reaction were observed. Cortisone (20 mg/kg for 6 days before challenge) significantly decreased cell accumulation. Delayed hypersensitivity was also elicited in the ear of sensitized animals. Extracts of sponges removed from antigen-challenged mice had macrophage chemotactic activity.

Synthesis and structure-activity relationship of spiro[isochromanpiperidine] analogues for inhibition of histamine release.

Two types of 1'-alkylspiro[isochroman-3,4-piperidines] and 1'-alkylspiro[isochroman-4,4'-piperidines] were prepared and examined for their biological activity. Several of the compounds inhibited the compound 48/80 induced histamine release from isolated rat peritoneal mast cells. The structural requirements for this activity in the present series are discussed.