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ABSTRACT: Neuromedin B (NMB) is a mammalian bombesin-like peptide that regulates exocrine/endocrine secretion, smooth muscle contraction, body temperature, and the proliferation of some cell types. Here we show that mRNA encoding NMB and its receptor NMBR are expressed in rat bone and brain tissues. Immunohistochemical analysis demonstrated that NMB and NMBR colocalize in osteoblasts, epiphyseal chondrocytes, and proliferative chondrocytes of growth plates from mouse hind limbs. Then, we investigated the effect of NMB on the proliferation of rat primary cultured osteoblasts. Proliferation assays and 5-bromo-2'-deoxyuridine (BrdU) incorporation assays demonstrated that NMB augments the cell number and enhances DNA synthesis in osteoblasts. Pretreatment with the NMBR antagonist BIM23127 inhibited NMB-induced cell proliferation and DNA synthesis. Western blot analysis showed that NMB activates extracellular signal-regulated kinase 1 or 2 (ERK1/2)/mitogen-activated protein kinase (MAPK) signaling in osteoblasts. Pretreatment with the MAPK/ERK kinase (MEK) inhibitor U0126 attenuated NMB-induced cell proliferation and DNA synthesis. We also investigated the effects of molecules that contribute to osteoblast proliferation and differentiation on Nmb expression in osteoblasts. Real-time PCR analysis demonstrated that 17β-estradiol (E2) and transforming growth factor β1 (TGFβ1) increase and decrease Nmb mRNA expression levels, respectively. Finally, proliferation assays revealed that the NMBR antagonist BIM23127 suppresses E2-induced osteoblast proliferation. These results suggest that NMB/NMBR signaling plays an autocrine or paracrine role in osteoblast proliferation, and contributes to the regulation of bone formation.
Journal of Endocrinology 02/2013; · 3.55 Impact Factor
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ABSTRACT: Neuromedin B (NMB), which was originally isolated from porcine spinal cord, is a mammalian bombesin-related peptide that exerts various physiological effects. Previously, we observed expression of NMB in rib cartilage from chicken. Here, we report the initial attempt to elucidate the role of NMB in cartilage. We used RT-PCR to measure the expression of NMB and its receptor (NMB-R) in mouse chondrogenic cell line ATDC5. During chondrogenic differentiation of ATDC5 cells, NMB mRNA transiently increased on day 4 and then decreased on day 14, whereas NMB-R mRNA decreased on days 7 and 14. We also characterized immunoreactive NMB in ATDC5 culture medium using a combination of specific radioimmunoassay (RIA) and reverse phase-high performance liquid chromatography (RP-HPLC). Furthermore, using the WST-8 assay, we demonstrated that NMB significantly induced ATDC5 proliferation; this was inhibited by NMB-R antagonist, BIM 23127. These results implicate that NMB is involved in cartilage development, either in an autocrine or paracrine manner.
Peptides 05/2012; 36(2):299-302. · 2.43 Impact Factor
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ABSTRACT: In addition to VPAC1 and VPAC2, PAC1 is involved in the pleiotropic action of pituitary adenylate cyclase activating polypeptide (PACAP) in the CNS. A luciferase reporter assay for the human PAC1 gene (-2160/+268) revealed that NGF treatment significantly augments the promoter activity of the PAC1 gene. Moreover, the Sp1 site at -282/-273 was shown to be essential for the NGF-augmented promoter activity of the PAC1 gene. Treatment with U0126, an MEK inhibitor, or Mithramycin A, an Sp1 inhibitor, significantly attenuated promoter activity. These results indicate that activation of Sp1 by the Ras/MAPK pathway might participate in neuron specific expression of the PAC1 gene.
FEBS letters 05/2012; 586(12):1731-5. · 3.54 Impact Factor
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ABSTRACT: N-arachidonoyl glycine (NAGly), a member of lipoamino acids, was reported to exhibit anti-inflammatory effects in experimental ear edema or peritonitis. However the underlying mechanisms have not been clarified so far. In this study, we attempt to investigate the effects of NAGly on macrophages, including the relevant signaling pathways. NAGly potently induced apoptosis in mouse macrophage-derived cell line, RAW264.7. Pretreatment with inhibitors for MEK and p38 MAPK prevented the apoptosis induced by NAGly, although NAGly activated ERK1/2, p38 MAPK and JNK. Further, we focused on implication of GPR18, one of the orphan G protein-coupled receptors, because NAGly has been reported as a candidate ligand for GPR18. Pretreatment with pertussis toxin or siRNA to knock down the expression of GPR18 significantly attenuated the apoptosis induced by NAGly. In mouse peritoneal macrophages, the expression of GPR18 mRNA was elevated in proinflammatory stimulated macrophages but not in anti-inflammatory stimulated macrophages; consistently, NAGly remarkably reduced cell viability of the former, as compared to the latter. These results suggest that NAGly might be involved in function of macrophages through GPR18.
Biochemical and Biophysical Research Communications 02/2012; 418(2):366-71. · 2.48 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides performing a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA region encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. To characterize the molecular forms of alternative splicing variants of PAC1, we examined the binding affinity and activation of two major second messenger pathways (cAMP production and changes in [Ca(2+)]( i )) by PACAP-27. Activation of cAMP was influenced by the variant in both of the EC1 domain and IC3 loops. In the N form in the EC1 domain, the suppressive effect of the HOP1 form in the IC3 loop was enhanced. Regarding the intracellular calcium mobilization assay, the rank order of the potency of PACAP-27 for the different PAC1 isoforms was S/HOP1>N/R~S/R>N/HOP1. In particular, PACAP-27 exhibited remarkable potency of calcium mobilization in the S/HOP1-expressing cells at sub-picomolar concentrations even though the affinities of PACAP-27 to the four PAC1 isoforms were not significantly different. This suggests the specific functions of PACAP-27 due to PACAP-27 preferring PAC1 activation, and leads in clarification of the pleiotoropic function of PACAP.
Journal of Molecular Neuroscience 11/2010; 42(3):341-8. · 2.50 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide localized in the testis at concentration comparable to that found in the brain, suggesting involvement in spermatogenesis. In this study, we identified the human PACAP testis-specific exon (TSE) 10.9 kb upstream from the translational start site and found that the testis-specific transcript of the human PACAP gene was found to be spliced from the TSE into a region of intron 2 without a frameshift. The resulting PACAP precursor has no signal peptide, suggesting that PACAP functions physiologically in an intracrine manner in the testis. The 5'-flanking region of the TSE contains an 80-bp fragment with potent promoter activity in testicular F9 cell. Electrophoresis mobility shift assays showed that proteins from the F9 nuclear extract interacted specifically with the 80-bp fragment. DNA affinity chromatography allowed isolation of the specific proteins bound to the 80-bp fragment, two of which were identified as Poly (ADP-ribose) polymerase-1 (PARP-1) and TIA-1-related protein (TIAR) by mass spectrometry. By using their siRNAs, the depletion of their proteins in F9 cells affected the potent promoter activity of the 80-bp fragment, suggesting that they might be involved in the testis-specific gene expression of PACAP.
Genes to Cells 06/2010; 15(6):595-606. · 2.68 Impact Factor
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ABSTRACT: The phosphodiesterase III inhibitor olprinone has been confirmed to improve myocardial function and increase cerebral blood flow; therefore, if olprinone exerts direct neuroprotective effects against global cerebral ischemia to the same degree as cilostazol, olprinone could be useful for cerebral resuscitation after cardiac arrest. We examined whether olprinone directly protected neuronal cells from global cerebral ischemia both in vivo and in vitro.
In a rat model of 10-minute global cerebral ischemia induced by 4-vessel occlusion, 0.3, 3, or 30 microg x kg(-1) x min(-1) olprinone or saline was infused for a periischemic period of 40 minutes (n = 6 for each group). Hippocampal CA1 neuronal cells were then counted 3 days after reperfusion, and the phosphorylation of cyclic adenosine 3'5'-monophosphate response element-binding protein was examined using Western blotting analyses of specimens obtained 15 minutes after reperfusion. In vitro, cultured cerebral neurons were exposed to 4 hours of hypoxia and glucose deprivation and then 24 hours of recovery in the absence or presence of olprinone (10(-11)-10(-5) mol x L(-1)). Cell viability was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Gaithersburg, MD).
In the rat model of global ischemia, the number of surviving CA1 neurons counted under a microscopic field in the 30 microg x kg(-1) x min(-1) olprinone-treated group (49.9 +/- 9.2) was significantly higher than that in the saline infusion control group (7.2 +/- 3.4), and olprinone treatment increased the phosphorylation of cyclic adenosine 3'5'-monophosphate response element-binding protein. The survival fraction of the neuronal cells cultured in the presence of olprinone was also significantly higher than that of cells cultured in the absence of olprinone in a dose-dependent manner.
Our study successfully demonstrated, for the first time, that olprinone had a protective effect on neuronal cells in vitro and in vivo, especially against global cerebral ischemia. These results suggest that olprinone might be useful for the treatment of patients experiencing global cerebral ischemia.
Anesthesia and analgesia 03/2010; 110(3):888-94. · 3.08 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neurotrophic factor through PAC1-R, PACAP-specific receptor, in the central nervous system. On the other hand, by interacting with nicotinic acetylcholine receptor (nAChR), nicotine exhibits several neuroprotective effects. Since the relevance of PACAP and nAChR signaling has not been reported so far, we attempted to investigate their relevance in terms of neuroprotection in PC12 cells. Regarding the effect of nicotine on PACAP gene expression, nicotine increased its mRNA level in time-dependent and dose-dependent manners in the PC12 cells differentiated with nerve growth factor (NGF). In addition, luciferase reporter assay demonstrated that nicotine treatment significantly augments the promoter activity of PACAP gene. Since PAC1-R mRNA expression was induced by NGF, PACAP exhibited neuroprotective effect against tunicamycin-induced cell death in the differentiated PC12 cells. Nicotine also exhibited the neuroprotective effect, which was significantly attenuated by Max.d.4 (PAC1-R specific antagonist). These results suggest that the increment of PACAP gene expression due to nicotine treatment might be involved in the neuroprotection by nicotine.
Journal of Molecular Neuroscience 08/2008; 36(1-3):73-8. · 2.50 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on downstream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors.
Molecular Pharmacology 08/2007; 72(1):103-11. · 4.88 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, exerts a variety of physiological functions through three types of G protein-coupled receptors, PAC1, VPAC1, and VAPC2. Characterization of the molecular forms of PAC1 in mouse heart revealed the presence of four types of variant receptors harboring the N or S variant in the first extracellular domain (EC1 domain) with or without the HOP1 insert in the third intracellular cytoplasmic loop (IC3 loop). Then, we assessed the binding affinity and ability to stimulate adenylyl cyclase of the PCA1 variant-expressing cells for PACAP. Adenylyl cyclase activation by PACAP was markedly influenced with the variant in the EC1 domain as well as that in the IC3 loop, in spite of a little difference in their binding properties. These data suggest that the combination of EC1 domain variants and IC3 loop variants might account for the diversity of intracellular signaling, which might contribute to multiple functions of PACAP including a role in the cardiovascular system.
Annals of the New York Academy of Sciences 08/2006; 1070:586-90. · 3.15 Impact Factor
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ABSTRACT: In rat-cultured astrocytes, pituitary adenylate cyclase-activating polypeptide (PACAP) activates gene expression and secretion of C-type natriuretic peptide (CNP) in a dose- and time-dependent manner. These results suggest that PACAP might be involved in the regulation of CNP biosynthesis in astrocytes.
Annals of the New York Academy of Sciences 08/2006; 1070:282-5. · 3.15 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactive neural elements have been detected in the mouse spinal cord. The discrepancy of PACAP actions in the role of sensory transmission has been proposed to have potentiation and inhibition on nociceptive responses after intrathecal application of PACAP. The aim of the present study was to assess nociceptive transmission of PACAP in the mouse spinal cord by comparison with that of substance P (SP). The intrathecal injection of PACAP induced licking or scratching behavior similar to that of SP. These PACAP-induced aversive behaviors showed different manner from SP-induced responses in point of time course. SP-induced aversive responses quickly increased and suddenly disappeared almost within 1 min. Meanwhile, following a long latency after the injection, PACAP-induced aversive responses gradually appeared, and then persisted more than 60 min. In the early phase, PACAP produced an increase of tail flick latency. Pretreatment with 6-hydroxydopamine (6-OHDA) which destroys noradrenaline neuron of descending pain inhibitory systems in the spinal cord markedly abridged the latency and augmented the duration of PACAP-induced aversive responses. In this way, PACAP exhibits diverse effects on nociception, such as an analgesic role in early phase of the injection and subsequently lasting algesia. These results suggest that PACAP as a neurotransmitter or neuromodulator might have crucial role in nociceptive transmission system.
Regulatory Peptides 01/2005; 123(1-3):117-22. · 2.11 Impact Factor
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ABSTRACT: Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5'-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.
Regulatory Peptides 01/2005; 123(1-3):9-14. · 2.11 Impact Factor
