Takashi Nakakura

Protective effect of resolvin E1 on the development of asthmatic airway inflammation.

Resolvin E1 (RvE1) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA), and strongly acts in the resolution of inflammation. We previously reported that RvE1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma. In the present study, to elucidate the effects of RvE1 on the development of asthmatic airway inflammation, we investigated whether RvE1 acts on different phases of an OVA-sensitized and -challenged mouse model of asthma. RvE1 treatments at the time of either OVA sensitization or at the time of OVA challenge were investigated and compared with RvE1 treatments at the time of both OVA sensitization and challenge. After RvE1 was administered to mice intraperitoneally at the time of both OVA sensitization and challenge, there were decreases in airway eosinophil and lymphocyte recruitment, as well as a reduction in Th2 cytokine and airway hyperresponsiveness. RvE1 treatment at the time of either OVA sensitization or challenge also improved AHR and airway inflammation. Our results suggest that RvE1 acts on several phases of asthmatic inflammation and may have anti-inflammatory effects on various cell types.

Each one of certain histidine residues in G-protein-coupled receptor GPR4 is critical for extracellular proton-induced stimulation of multiple G-protein-signaling pathways

GPR4, previously proposed as the receptor for sphingosylphosphorylcholine, has recently been identified as the proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways, including the G(s)-protein/cAMP, G(12/13)-protein/Rho, and G(q)-protein/phospholipase C pathways. In the present study, we examined whether extracellularly located histidine residues of GPR4 sense extracellular protons and, if so, whether a certain histidine residue is critical for coupling to the single or multiple signaling pathway(s). We found that the mutation of histidine residue at 79, 165, or 269 from the N-terminal of GPR4 to phenylalanine shifted the half-maximal effective concentration (EC(50)) of proton-induced signaling activities to the right, including cAMP accumulation, SRE promoter activity reflecting Rho activity, and NFAT promoter activity reflecting phospholipase C signaling activity, without an appreciable change in the maximal activities. These results suggest that the protonation of each one of histidine residues at 79, 165, and 269 in GPR4 may be critical for conformational change of the receptor for coupling to multiple intracellular signaling pathways through G-proteins.

Neuroendocrine regulation of thyroid-stimulating hormone secretion in amphibians

The hypothalamic peptides thyrotropin‐releasing hormone (TRH), gonadotropin‐releasing hormone (GnRH), and corticotropin‐releasing factor (CRF), which have been postulated as acting as thyroid‐stimulating hormone (TSH)‐releasing hormone in amphibians, were tested for their activity by employing a recently developed radioimmunoassay for bullfrog (Rana catesbeiana) TSH. CRF markedly stimulated the release of TSH from both adult and larval bullfrog pituitary cells. Both TRH and GnRH moderately stimulated the release of TSH from adult pituitary cells but not from larval ones. The release of TSH was also enhanced by bullfrog hypothalamic extracts. The hypothalamic extract‐evoked release of TSH was markedly reduced by a CRF receptor antagonist, suggesting that CRF and/or CRF‐related peptides are the main TSH‐releasing factors occurring in the bullfrog hypothalamus. Experiments using CRF receptor agonists and antagonists revealed that CRF acts through the type 2 receptor. With regard to other hypothalamic substances that influence the release of TSH, pituitary adenylate cyclase‐activating polypeptide and vasoactive intestinal polypeptide were found to be potent stimulators and somatostatin an inhibitor of TSH release. Thus, it becomes clear that the main regulatory peptides controlling TSH secretion in amphibians are different from those in mammals. Triiodothyronine did not affect the basal release of TSH from the pituitary of either larval or adult bullfrogs but suppressed the CRF‐induced release of TSH, suggesting that negative feedback by thyroid hormone is functioning both in larvae and adults.

Inhibition of superoxide anion production by extracellular acidification in neutrophils.

Extracellular acidification inhibited formyl-Met-Leu-Phe- or C5a-induced superoxide anion (O(2)(-)) production in differentiated HL-60 neutrophil-like cells and human neutrophils. A cAMP-increasing agonist, prostaglandin E(1), also inhibited the formyl peptide-induced O(2)(-) production. The inhibitory action on the O(2)(-) production by extracellular acidic pH was associated with cAMP accumulation and partly attenuated by H89, a protein kinase A inhibitor. A significant amount of mRNAs for T-cell death-associated gene 8 (TDAG8) and other proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-family receptors is expressed in these cells. These results suggest that cAMP/protein kinase A, possibly through proton-sensing G-protein-coupled receptors, may be involved in extracellular acidic pH-induced inhibition of O(2)(-) production.

Deficiency of Proton-Sensing Ovarian Cancer G Protein-Coupled Receptor 1 Attenuates Glucose-Stimulated Insulin Secretion

Ovarian cancer G protein-coupled receptor 1 (OGR1) has been shown as a receptor for protons. In the present study, we aimed to know whether OGR1 plays a role in insulin secretion and, if so, the manner in which it does. To this end, we created OGR1-deficient mice and examined insulin secretion activity in vivo and in vitro. OGR1 deficiency reduced insulin secretion induced by glucose administered ip, although it was not associated with glucose intolerance in vivo. Increased insulin sensitivity and reduced plasma glucagon level may explain, in part, the unusual normal glucose tolerance. In vitro islet experiments revealed that glucose-stimulated insulin secretion was dependent on extracellular pH and sensitive to OGR1; insulin secretion at pH 7.4 to 7.0, but not 8.0, was significantly suppressed by OGR1 deficiency and inhibition of G(q/11) proteins. Insulin secretion induced by KCl and tolbutamide was also significantly inhibited, whereas that induced by several insulin secretagogues, including vasopressin, a glucagon-like peptide 1 receptor agonist, and forskolin, was not suppressed by OGR1 deficiency. The inhibition of insulin secretion was associated with the reduction of glucose-induced increase in intracellular Ca(2+) concentration. In conclusion, the OGR1/G(q/11) protein pathway is activated by extracellular protons existing under the physiological extracellular pH of 7.4 and further stimulated by acidification, resulting in the enhancement of insulin secretion in response to high glucose concentrations and KCl.

The elongation of primary cilia via the acetylation of α-tubulin by the treatment with lithium chloride in human fibroblast KD cells

Primary cilium, an organelle found on nearly every cell in the human body, typically serves as the mechanical sensor of the cell. Lithium ion is known to promote the elongation of primary cilia in a variety of cell types, but it is unknown whether lithium is involved in the acetylation of α-tubulin which is essential for the assembly of primary cilia. In order to reveal the relationship between the elongation of primary cilia with lithium and the acetylation of α-tubulin, we first observed the formation and structure of primary cilia in KD cells, a cell line deriving fibroblasts in human labium. Subsequently, by immunohistochemical and western blot analysis we elucidated that the length of primary cilia and acetylation of α-tubulin are regulated by lithium chloride (LiCl) in the medium in a time- and concentration-dependent manner. We next performed the RT-PCR, RNAi-based experiments and biochemical study using an inhibitor of glycogen synthase kinase-3βGSK-3β). We found that LiCl mobilizes the α-tubulin N-acetyltransferase 1 (αTAT1) in the signaling pathway mediating GSK-3β and adenylate cyclase III. In conclusion, our results suggested that LiCl treatments activate αTAT1 by the inhibition of GSK-3β and promote the α-tubulin acetylation, and then elongate the primary cilia.

Sinusoid development and morphogenesis may be stimulated by VEGF-Flk-1 signaling during fetal mouse liver development.

Early morphogenesis of hepatic sinusoids was histochemically and experimentally analyzed, and the importance of VEGF‐Flk‐1 signaling in the vascular development was examined during murine liver organogenesis. FITC‐gelatin injection experiments into young murine fetuses demonstrated that all primitive sinusoidal structures were confluent with portal and central veins, suggesting that hepatic vessel development may occur via angiogenesis. At 12.5–14.5 days of gestation, VEGF receptors designated Flk‐1, especially their mature form, were highly expressed in endothelial cells of primitive sinusoidal structures and highly phosphorylated on their tyrosine residues. At the same time, VEGF was also detected in hepatoblasts/hepatocytes, hemopoietic cells, and megakaryocytes of the whole liver parenchyma. Furthermore, the addition of VEGF to E12.5 liver cell cultures significantly induced the growth and branching morphogenesis of sinusoidal endothelial cells. Therefore, VEGF‐Flk‐1 signaling may play an important role in the growth and morphogenesis of primitive sinusoids during fetal liver development. Developmental Dynamics 239:386–397, 2010.

The spatial and temporal expression of delta-like protein 1 in the rat pituitary gland during development

An analysis of secreted proteins by the signal sequence trap method using a cDNA library of the rat pituitary anlage at embryonic days (E) 13.5 revealed the abundant expression of delta-like protein 1 (Dlk1) in the pituitary gland. Dlk1, an epidermal growth factor-like repeat protein in preadipocytes, functions in maintaining the preadipose state. Expression of Dlk1 mRNA in the pituitary at E13.5 and in the adult pituitary was confirmed by in situ hybridization. The expression pattern of Dlk1 during pituitary development was also studied by immunohistochemistry. Dlk1 protein first appeared in Rathke’s pouch and the infundibulum at E11.5; as development proceeded, expression became restricted to the pars distalis and pars tuberalis (PT). Dlk1 was expressed in most ACTH cells during the embryonic stages, but its expression was limited to only a few ACTH cells in the adult pituitary. It was also expressed in a small population of TSH, GTH, and PRL cells throughout development, whereas it was present in the cytoplasm of most GH cells at all developmental stages. Similarly, Dlk1 was localized in the cytoplasm of PT cells during development. These findings provide new insights into the mechanism of Dlk1 regarding its regulation of pituitary hormone-secreting cells during development.

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.

Gene expression of vascular endothelial growth factor-A in the pituitary during formation of the vascular system in the hypothalamic-pituitary axis of the rat

Techniques involving fluorescein-5-isothiocyanate-conjugated gelatin injection, immunohistochemistry, and in situ reverse transcription/polymerase chain reaction (RT-PCR) revealed a close relationship between vascular endothelial growth factor (VEGF)-A-expressing cells and microvessels in the hypothalamic-pituitary axis of the rat. In situ RT-PCR clearly indicated the presence of VEGF-A mRNA-expressing cells in the pars tuberalis and in the pars distalis both at embryonic day 15.5 (E15.5) and in later developmental stages. The primary capillaries extended along the developing pars tuberalis, whereas the portal vessels penetrated into the pars distalis at E15.5 and subsequently expanded into the lobe to connect with the secondary capillary plexus, emerging in the pars distalis. At the same time, several VEGF-A-positive cells appeared in the pars distalis. These VEGF-A-positive cells were found to correspond to a portion of adrenocorticotropin (ACTH) cells by dual-staining for in situ RT-PCR and immunohistochemistry, suggesting that some ACTH cells have the potential to produce VEGF-A. Thus, the present study suggests that VEGF-A is involved in the development of the primary capillaries and in the vascularization of the pars distalis, but not in the portal vessels since the formation of portal vessels begins at E13.5, before the appearance of VEGF-A in the rostral region of the pars distalis.