Dysfunction of the hypothalamic-pituitary-adrenal axis in STX1A knockout mice.

HPC-1/syntaxin1A (STX1A) is considered to regulate exocytosis in neurones and endocrine cells. Previously, we reported that STX1A null mutant (STX1A KO) mice unexpectedly showed normal glutamatergic and GABAergic fast synaptic transmission but exhibited disturbances in monoaminergic transmission, such as serotonin, 5-hydroxytryptamine (5-HT), which may induce attenuation of latent inhibition. These results suggest that STX1A may contribute to dense-core vesicle exocytosis in vivo. Thus, we hypothesised that the lack of STX1A might affect the secretion of several hormones, as also mediated by dense-core vesicles exocytosis. In the present study, we focused on the hypothalamic-pituitary-adrenal (HPA) axis, which is a neuroendocrine system that regulates responses to stress stimuli and is considered to be associated with neuropsychiatric disorders. Specifically, we examined whether the HPA axis is impaired in STX1A KO mice. Interestingly, plasma concentrations of both corticosterone (CORT) and adrenocorticotrophin hormone (ACTH) during the resting condition decreased in STX1A KO mice compared to WT mice. Additionally, elevated plasma CORT, ACTH and corticotrophin-releasing hormone (CRH) which were usually observed after acute restraint stress, were also reduced in STX1A KO mice. We also observed the suppression of 5-HT-induced CRH release in STX1A KO mice in vitro. Furthermore, an in vivo microdialysis study revealed that the elevation of extracellular 5-HT in the hypothalamus, which was induced by the selective serotonin reuptake inhibitor, fluoxetine, was significantly reduced in STX1A KO mice compared to WT mice. 5-HT elevation in the hypothalamus, which was induced by acute restraint stress, was also reduced in STX1A KO mice. Finally, STX1A KO mice showed abnormal behavioural responses after mild restraint stress. These results indicate that the lack of STX1A could induce dysfunction of the HPA axis, and the deficit may result in abnormal behavioural properties, such as unusual responses to stress stimuli.

Enhancement of synaptic transmission and nociceptive behaviour in HPC-1/syntaxin 1A knockout mice following peripheral nerve injury.

Our previous analysis of HPC-1/syntaxin 1A knockout (KO) mice indicated that HPC-1/syntaxin 1A plays an important role in the synaptic plasticity of the hippocampus in vitro and learning behaviour in vivo. In order to gain further insights into the physiological functions of HPC-1/syntaxin 1A, we studied the changes in the plasticity of synaptic transmission in the superficial dorsal horn of the spinal cord following a peripheral nerve injury in HPC-1/syntaxin 1A KO and wild-type (WT) mice. The von Frey filament test revealed that partial ligation of the sciatic nerve caused neuropathic pain in both WT and KO mice. However, KO mice showed significant enhancement of mechanical allodynia as compared with WT mice. Tight-seal whole-cell recordings were obtained from neurons in the superficial dorsal horn of the spinal cord slices. Electrical stimulus-evoked excitatory postsynaptic currents (EPSCs), asynchronous EPSCs (aEPSCs) in the presence of strontium, and spontaneously occurring miniature EPSCs (mEPSCs) were analysed. Prior to peripheral nerve ligation, no significant differences were observed in the properties of evoked EPSCs, aEPSCs and mEPSCs in KO and WT mice. Seven-14 days after partial ligation, the amplitude of evoked EPSCs and the frequency of aEPSCs and mEPSCs in KO mice were significantly greater than those in WT mice; however, the amplitude of aEPSCs and mEPSCs remained unchanged in both groups. Enhanced allodynia behaviour and significant enhancement of excitatory synaptic transmission following peripheral nerve ligation in KO mice suggest that HPC-1/syntaxin 1A might play a role in synaptic plasticity in the nociceptive pathway.

Expression of the exocytotic protein syntaxin in mouse oocytes.

Syntaxin is an integral membrane protein that is involved in membrane fusion. The exocytosis of the contents of cortical granules, secretory vesicles located in the cortex of an egg, modify the extracellular environment to block additional spermatozoa from penetrating the newly fertilized egg. The aim of this study was to characterize syntaxin expression in mouse oocytes, and to determine the specific isoform that is expressed. Syntaxin was demonstrated in the mouse ovary and in mouse oocytes by both western blot and reverse transcription-polymerase chain reaction analyses. Syntaxin 4 was specifically expressed in metaphase II oocytes. Syntaxin was also immunolocalized within metaphase II oocytes and one-cell embryos with pronuclei using laser scanning confocal microscopy. In metaphase II oocytes, syntaxin was located on the plasma membrane and in the cortex, where cortical granules are present, but was not seen at sites free of cortical granules. In one-cell embryos, no cytoplasmic region was free of syntaxin immunoreactivity. Immunoelectron microscopy detected syntaxin on both the plasma membrane and the vesicle membranes in mouse metaphase II oocytes. In conclusion the results indicate that syntaxin 4 co-localizes with cortical granules and participates in membrane fusion and exocytosis during the cortical reaction.

Optical recording of spontaneous respiratory neuron activity in the rat brain stem.

We report on the optical imaging of spontaneous respiratory neuron bursts in the ventrolateral medulla (VLM) of medullary slices or brain stem-spinal cord preparations. A medullary slice with a thickness of 1.0-1.4 mm or brain stem-spinal cord from 0- to 4-d-old rats was stained with fluorescent voltage-sensitive dye, RH795. Optical signals were recorded as a fluorescence change by using an optical recording apparatus with a 128 x 128 photodiode array and a maximum time resolution of 0.6 ms. Motoneuronal activity was simultaneously recorded at the hypoglossal nerve roots or fourth cervical ventral roots. Fluorescence changes corresponding to the spontaneous inspiratory burst activity were detected in the hypoglossal nucleus and VLM in slice preparations, and in a limited area extending rostrocaudally in the VLM of the brain stem-spinal cord preparation. These measurements did not require signal averaging by multiple trials. Results suggest that inspiratory neurons are localized in more compact form at the level of the nucleus ambiguous than at the more rostral VLM, and that peak activity during the inspiratory phase propagates from the caudal to the rostral VLM. In 60% of brain stem-spinal cord preparations, weak and scattered fluorescence changes preceding the inspiratory burst activity were detected more predominantly in the rostral part of the VLM. The present findings show the feasibility of optical recordings for the in vitro analysis of spontaneous respiratory neuron activity in the medulla.

Roles of the cytoplasmic and transmembrane domains of syntaxins in intracellular localization and trafficking.

Syntaxins are target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) involved in docking and fusion of vesicles in exocytosis and endocytosis. Many syntaxin isoforms have been isolated, and each one displays a distinct intracellular localization pattern. However, the signals that drive the specific intracellular localization of syntaxins are poorly understood. In this study, we used indirect immunofluorescence analysis to examine the localization of syntaxin chimeras, each containing a syntaxin transmembrane domain fused to a cytoplasmic domain derived from a different syntaxin. We show that the cytoplasmic domains of syntaxins 5, 6, 7 and 8 have important effects on intracellular localization. We also demonstrate that the transmembrane domain of syntaxin 5 is sufficient to localize the chimera to the compartment expected for wild-type syntaxin 5. Additionally, we find that syntaxins 6, 7 and 8, but not syntaxin 5, are present at the plasma membrane, and that these syntaxins cycle through the plasma membrane by virtue of their cytoplasmic domains. Finally, we find that di-leucine-based motifs in the cytoplasmic domains of syntaxins 7 and 8 are necessary for their intracellular localization and trafficking via distinct transport pathways. Combined, these results suggest that both the cytoplasmic and the transmembrane domains play important roles in intracellular localization and trafficking of syntaxins.

IL-10 inhibits granulocyte-macrophage colony-stimulating factor-dependent human monocyte survival at the early stage of the culture and inhibits the generation of macrophages.

We previously demonstrated that IL-10 alone does not stimulate growth and differentiation of human monocytes, but enhances those of monocytes stimulated with M-CSF. We studied here the effect of IL-10 on human monocytes stimulated with GM-CSF. Monocytes stimulated with GM-CSF alone survived and developed into macrophages. Monocytes cultured with GM-CSF plus IL-10, however, died through apoptosis. IL-10 decreased expression of bcl-2, bcl-x(L), and mcl-1- but not bax mRNA in monocytes stimulated with GM-CSF. IL-10 did not change the expression of mRNA of both GM-CSFR alpha-chain and beta-chain, but inhibited tyrosine phosphorylation of STAT5 and extracellular signal-regulated kinases 1 and 2 in the monocytes. The inhibitory effect of IL-10 was restricted to treatment 48 h after stimulation with GM-CSF. Addition of IL-10 after that time induced neither apoptosis nor a decrease in expression of bcl-2, bcl-x(L), and mcl-1 mRNA. IL-10, however, inhibited LPS-induced TNF-alpha production even in these cells, indicating that the cells still possessed responsiveness to IL-10. Monocytes pretreated for >48 h with GM-CSF became resistant to GM-CSF withdrawal, and the cells could survive without GM-CSF. These results indicate that IL-10 selectively inhibits GM-CSF-dependent monocyte survival by inhibiting the signaling events induced by GM-CSF, but the timing of addition of IL-10 is critical, and IL-10 had to be added within 48 h after stimulation with GM-CSF to achieve the inhibitory effect. These results taken together with our previous results indicate that IL-10 plays a pivotal role in monocyte survival and development into macrophages in concert with M-CSF and GM-CSF.

Increase in number of functional release sites by cyclic AMP-dependent protein kinase in cultured neurons isolated from hippocampal dentate gyrus.

The enhancement of synaptic exocytosis is one form of long-term potentiation (LTP) of synaptic transmission. As possible mechanisms underlying this enhancement, increases in the release probability and/or the number of release sites are suggested. To obtain direct evidence for the increase in the number of functional release sites induced by protein kinase A (PKA) cascade, we attempted to visualize functional release sites using styryl dyes, FM4-64 and FM1-43, and investigated the effects of PKA on the release sites. A PKA activator FSK increased the number of active release sites by approximately 20-30%. A direct PKA activator, Sp-cAMPS, showed the same effect, which was blocked by a PKA inhibitor, KT5720, suggesting that this effect was mediated by PKA. This PKA-dependent increase in the number of release sites requires Ca(2+) in the bath solution, and Sr(2+) can not be a substitute for Ca(2+). Since the number of functional release sites is approximately half the total number of synaptophysin-immunoreactive sites, the PKA dependent activation of silent release sites of DG neuron terminals is suggested.

Suppressive mechanisms of EPA on human T cell proliferation.

In vivo and in vitro experiments show that polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) inhibit mitogen- or antigen-stimulated proliferation of T cells in rodents and humans. However, the exact manner and mechanisms by which PUFA inhibits T cell proliferation is not clear. In the present study, we investigated the suppressive effects of EPA, an n-3 PUFA, on PHA stimulated human peripheral blood T cells. Our results showed that EPA suppresses mitogen- or antigen-stimulated human T cell proliferation by at least 2 steps; step 1) EPA suppresses T cell proliferation by inhibiting IL-2R alpha expression and IL-2 production; step 2) EPA induces cell death of blast T cells without reducing the expression of IL-2R alpha. We also showed that EPA selectively stimulates the cell death of blast T cells but not resting T cells. The suppressive effect of EPA was mediated via the production of reactive oxygen products, because EPA-stimulated H2O2 production and the suppressive effect of EPA was restored by addition of catalase or NAC. These results taken together suggest that such immunosuppressive effects of EPA may explain the apparent benefits of EPA-enriched diets for patients with inflammatory disorders.

Suppression of transmitter release by Tat HPC-1/syntaxin 1A fusion protein.

It has been reported that the fusion protein with the protein transduction domain (PTD) peptide of HIV-1 Tat protein can be internalized through the cell membrane of intact cells, although the exact mechanism is unknown. In this report, we investigated whether this new method could be used for the molecular analysis of exocytosis via HPC-1/syntaxin 1A, which plays an important role in transmitter release. When applied to PC12 cells, Tat PTD fusion proteins were rapidly internalized into most cells. In order to show that the internalized protein remained biologically active, the H3 domain of HPC-1/syntaxin 1A was fused to Tat PTD (Tat-H3). Transmitter release in PC12 cells was suppressed by Tat-H3 treatment. These results indicate that the Tat fusion protein is a useful tool for analyzing the process of transmitter release.

Altered immunoreactivity of HPC-1/syntaxin 1A in proliferated nerve fibers in the human aganglionic colon of Hirschsprung's disease.

To clarify the pathogenesis of excessive proliferation of extrinsic nerve fibers in the aganglionic colon of patients with Hirschsprung's disease (HD), we immunohistochemically determined the role that exocytosis-related proteins play in the regulation of exocytosis using the antibody to HPC-1/syntaxin 1A, an exocytosis-related protein. Localization of exocytosis-related proteins (HPC-1/syntaxin 1A, N-ethylmalemide-sensitive fusion protein (NSF), soluble NSF attachment protein (SNAP), synaptotagmin, synaptobrevin, and synaptosome-associated protein 25 (SNAP-25)) was determined in surgical specimens obtained from normal proximal and aganglionic distal segments of the colon of 7 infant patients with HD. In the normal ganglionic colon, Auerbach's plexus, Meisner's plexus, nerve fibers in the muscle layer, and ganglion cells were immunopositive for all six kinds of antisera. In the aganglionic segments, numerous proliferated nerve fibers and hypertrophied nerve bundles were detected in the submucosal layer and myenteric layer by NSF, SNAP, synaptotagmin, synaptobrevin, and SNAP-25. However, HPC-1/syntaxin 1A was not recognized in the proliferated nerve fibers of the submucosal layer or the hypertrophied nerve bundles of the aganglionic segment. These findings show that immunoreactivity of HPC-1/syntaxin 1A was decreased in the affected bowel segments of patients with HD and may be related to the pathogenesis of extrinsic nerve-fiber proliferation in the aganglionic colon of HD.

Human alveolar macrophages and granulocyte-macrophage colony-stimulating factor-induced monocyte-derived macrophages are resistant to H2O2 via their high basal and inducible levels of catalase activity.

Human alveolar macrophages (A-MPhi) and macrophages (MPhi) generated from human monocytes under the influence of granulocyte-macrophage colony-stimulating factors (GM-MPhi) express high levels of catalase activity and are highly resistant to H(2)O(2). In contrast, MPhi generated from monocytes by macrophage colony-stimulating factors (M-MPhi) express low catalase activity and are about 50-fold more sensitive to H(2)O(2) than GM-MPhi or A-MPhi. Both A-MPhi and GM-MPhi but not M-MPhi can induce catalase expression in both protein and mRNA levels when stimulated with H(2)O(2) or zymosan. M-MPhi but not GM-MPhi produce a large amount of H(2)O(2) in response to zymosan or heat-killed Staphylococcus aureus. These findings indicate that GM-MPhi and A-MPhi but not M-MPhi are strong scavengers of H(2)O(2) via the high basal level of catalase activity and a marked ability of catalase induction and that catalase activity of MPhi is regulated by colony-stimulating factors during differentiation.

Interleukin-10 contributes development of macrophage suppressor activities by macrophage colony-stimulating factor, but not by granulocyte-macrophage colony-stimulating factor.

Macrophages are known to possess suppressor activities in immune responses. To determine the effects of GM-CSF and M-CSF on the expression of macrophage suppressor activities, monocyte-derived macrophages cultured with GM-CSF (GM-Mphis) were compared with those cultured with M-CSF (M-Mphis) for antigen-specific proliferation and interferon-gamma (IFN-gamma) production by lymphocytes. Both GM-Mphis and M-Mphis equally suppressed lymphocyte proliferation, but only M-Mphis suppressed IFN-gamma production in response to purified protein derivative (PPD). M-Mphis, but not GM-Mphis, released IL-10 not only in the course of macrophage differentiation but also in response to PPD after maturation to macrophages. From the results that (i) exogenous IL-10 suppressed IFN-gamma production, but not proliferation of lymphocytes, and that (ii) neutralizing antibody to IL-10 reversed suppressor activities of M-Mphis on IFN-gamma production, but not lymphocyte proliferation, it appeared that IL-10 was the major factor responsible for suppression of IFN-gamma production. Thus, these results suggest that only M-CSF augments IL-10-dependent suppressor activity of macrophages on IFN-gamma production and that both GM-CSF and M-CSF induce IL-10-independent macrophage suppressor activity on lymphocyte proliferation.

Molecular cloning of guinea pig angiotensin type 1 receptor.

The primary structure of cDNA encoding of the angiotensin type 1 receptor (AT(1)R) was cloned from guinea pig liver. Guinea pig AT(1)R (GP-AT(1)R) cDNA clone contains a 1,077-bp open reading frame which encodes a protein consisting of 359 amino acid residues. GP-AT(1)R amino acid sequence showed a 92% level of identity among mammalian species. GP-AT(1)R is expressed in liver, kidney, adrenal gland, heart and colon.

Ornithine-containing lipids stimulate CD14-dependent TNF-alpha production from murine macrophage-like J774.1 and RAW 264.7 cells.

The ornithine-containing lipids (OL)-induced cytokine production pattern in macrophage-like J774.1 and RAW 264.7 cells was different from that in the peritoneal macrophages previously reported. OLs, as well as lipopolysaccharide (LPS) of Escherichia coli, strongly induced tumor necrosis factor (TNF) alpha but not interleukin (IL)-1beta in J774.1 cells. In the RAW cells, IL-1beta, TNF-alpha and prostaglandin E(2) were strongly induced by the OLs and LPS. OL- and serine-glycine-containing lipid (SGL)-induced TNF-alpha production in J774.1 and RAW 264.7 cells required serum. However, in CD14-deficient LR-9 cells, TNF-alpha was not induced by the OLs in the presence or absence of serum. OLs and a SGL almost completely inhibited the binding of (125)I-LPS to J774.1 cells. These results suggested that OLs and SGL activate macrophages via the CD14-dependent pathway.

Transcriptional regulation of HIV-1 LTR during antigen-dependent activation of primary T cells by dendritic cells.

Numerous factors are known to bind human immunodeficiency virus (HIV) long terminal repeat (LTR) and activate viral transcription, but little is known as to how they function in naturally activated T cells and to what extent their binding is relevant to HIV replication in vivo. To characterize the HIV LTR-binding factors responsible for antigen-dependent activation of HIV, we examined replication of LTR mutant viruses in CD4+ T cells activated by different stimuli. NF-kappaB or Sp1 mutant virus replicated well in CD4+ T cells activated by phorbol ester and calcium ionophore. When they were activated by antigen-pulsed dendritic cells, the replication of the Sp1-deleted virus was severely impaired in CD45RA+, but not in CD45RO+ T cell subsets that dominantly produce interleukin-2 (IL-2). Stimulation via CD3/CD28 induced a high level of IL-2 production in both T cell subsets, but Sp1-deleted virus poorly replicated in CD45RA+ subset. The level of NF-kappaB and Sp1-binding factors did not differ between these subsets. Our results suggest that additional cofactors distinct from IL-2-inducing signaling molecules are important for LTR activation during antigen-dependent T cell activation.

Syntaxin 1A is expressed in airway epithelial cells, where it modulates CFTR Cl(-) currents.

The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble syntaxin 1A peptides and by the syntaxin 1A binding protein, Munc-18. In the present study, we determined whether syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-syntaxin 1A interaction, including soluble syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.

HPC-1/syntaxin 1A suppresses exocytosis of PC12 cells.

The membrane protein syntaxin (originally named HPC-1) is involved in vesicle trafficking and required for neurotransmitter release at nerve terminals. The presence of syntaxin on target membranes is hypothesized to confer specificity to targeting and fusion via interactions with complementary vesicle-associated proteins. To elucidate the function of syntaxin 1A in exocytosis, HPC-1/syntaxin 1A-reduced PC12h cells (PC12h/Deltasyx) that were stably transfected with a plasmid for antisense syntaxin 1A expression were constructed. Depolarizing stimulation of PC12h/Deltasyx enhanced dopamine release, compared with PC12h. There was a strong inverse correlation between syntaxin 1A protein expression and enhancement of dopamine release. Reduction of syntaxin 1A had no effect on increase of the cytoplasmic free Ca2+ concentration by depolarized stimulation. Moreover, PC12h/Deltasyx clones similarly enhanced of exocytosis by native secretagogues. These results indicate that syntaxin 1A has more than one function in exocytosis.

A typical bacterial ornithine-containing lipid Nalpha-(D)-[3-(hexadecanoyloxy)hexadecanoyl]-ornithine is a strong stimulant for macrophages and a useful adjuvant.

Nalpha-[3-(Hexadecanoyloxy)hexadecanoyl]-ornithine is a typical bacterial ornithine-containing lipid (OL). The configuration of the 3-hydroxy fatty acids in the OL was proved to be D by using HPLC with chiral column. For this analysis, Nalpha-(D or L)-[3-(hexadecanoyloxy)hexadecanoyl]-L-ornithine were synthesized and used as standards. The typical bacterial OL, as well as the synthesized one, exhibited strong interleukin-1- and prostaglandin E2-inducing activities, and further, it induced the production of high IgG anti-tetanus toxoid antibodies in mice. The typical OL is expected to be utilized as a nontoxic, potent adjuvant.

Inhibition of microtubule assembly by HPC-1/syntaxin 1A, an exocytosis relating protein.

HPC-1/syntaxin 1A (HPC-1), which has been identified as a presynaptic membrane protein, is believed to regulate the synaptic exocytosis as a component of t-SNARE. The distribution of the protein, however, is not restricted to the synaptic terminal, but it has been found to locate on the axonal membrane. When the expression of HPC-1 was suppressed, neurite sprouting was enhanced in cultured neurons. These findings suggest that HPC-1 possesses other functions than the regulation of the membrane fusion in neurotransmitter release. Rather it may also participate in the morphogenesis of neurons through membrane fusion, and possibly through cytoskeleton. HPC-1 has a sequence resemble to the assembly promoting sequence of heat stable MAPs in residues 89-106, suggesting that it can bind tubulin and be involved in microtubule system. Thus, both the tubulin binding property and the effect on microtubule assembly of HPC-1 were examined in vitro using a mutated HPC-1 lacking the C-terminal transmembrane region (HPC-deltaTM), which was overexpressed in E. coli. Affinity column chromatography showed that tubulin was found to bind HPC-1 directly. Synthetic peptide which corresponds to the residues 89-106 competitively inhibited the tubulin-HPC-1 binding, indicating that the sequence is responsible for the tubulin binding. In addition, chemical cross-linking with EDC revealed that one HPC-1 molecule can bind per one monomeric tubulin molecule. Light scattering measurement of microtubule polymerization showed that HPC-1 decreased the rate of the pure tubulin polymerization. Direct observation of single microtubules under dark-field microscopy showed that the growth rate of microtubule decreased by HPC-1. After shortening stopped, microtubules often spent attenuate phases, in which neither growing nor shortening was detected. When another mutant HPC-1 which is composed of residues 1-97 and lacks tubulin binding activity was used, however, the suppression of microtubule polymerization was not observed. These results suggest that HPC-1 is a potent regulator of microtubule polymerization, which directly bind tubulin subunit and decrease the polymerization activity.