• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.109-115
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• 2005

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughoutthe lifetime. In this study, we analyzed the expression of receptors of $P2Y_{10}$, purinergic receptor families in murine hematopoietic stem cells, hematopoietic progenitor cells. In addition, the biological activity of $P2Y_{10}$ was investigated with B lymphocyte cell line, Ba/F3 in effect to cell growth and cell cycle. From the analysis of expression in hematopoieticstem cell. and progenitor with RT-PCR, $P2Y_{10}$ was strongly expressed in murine hematopoieticstem cells (c-kit+ Sca-l+ Lin-) and progenitor cell population, such as c-kit- Sca-l+ Lin-, c-kit+ Sca-l- Lin- and c-kit- Sca-l- Lin-. To investigate the biological effects by $P2Y_{10}$, retroviral vector from subcloned murine $P2Y_{10}$ cDNA was used fur gene introduction into Ba/F3 cells, and stable transfectant cells were obtained by flow cytometry sorting. In cell proliferation assay, the proliferation ability of $P2Y_{10}$ receptor gene­transfected cells was strongly inhibited, and the cell cycle was arrested at G1 phase. These result suggest that the $P2Y_{10}$ may be involved the biological activity in hematopoietic stem cells and immature B lymphocytes.

• Korean Journal of Veterinary Service
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• v.19 no.2
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• pp.154-162
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• 1996

The effcets of adenosine 5'-triphosphate(ATP) were investigated on the uterine smooth muscle motility in the pig. The results were summarized as follows: 1. The effects of the porcine uterine smooth muscle and the contractile responses increased between the concentration of ATP $10^{-5}$ and $10^{-3}$ M with a dose-dependent manner. 2. The contractile response induced by ATP($10^{-4}$ M) was not blocked by pretreatment with cholinergic receptor blocker, atropine ($10^{-6}$ M) 3. The contractile response induced by ATP ($10^{-4}$ M) was not blocked by pretreatment with $\alpha$ -adrenergic receptor blocker, phentolamine(10$^{-6}$ M) and ${\beta}$-adrenergic blocker, propranolol ($10^{-6}$ M). 4. The contractile response induced by ATP($10^{-4}$ M) was not appeared in 4Ca^{++}$ -free medium. As the concentration of $Ca^{++}$ in $Ca^{++}$ -free medium was increased, the contractile response induced by ATP ($10^{-4}$ M) was enhenced but was completely inhibited by pretreatment with $Ca^{++}$ -channel blocker, papaverine($10^{-6}$ M) or verapamil($10^{-6}$ M). From these results, it was conclued that the effects of ATP were the contraction mediated by purinergic receptor in uterine smooth muscle of pig.

• Journal of Yeungnam Medical Science
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• v.12 no.2
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• pp.246-259
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• 1995

This study aimed to investigate the mechanism of action of baclofen on the detrusor muscle isolated from rat. Rats (Sprague-Dawley) were sacrificed by decapitation and exsanguination. Horizontal muscle strips of $2mm{\times}15mm$ were prepared for isometric myography in isolated muscle chamber bubbled with 95% / 5%-$O_2$ / $CO_2$ at $37^{\circ}C$, and the pH was maintained at 7.4. Detrusor strips contracted responding to the electrical field stimulation (EFS) by 2 Hz, 20 msec, monophasic square wave of 60 VDC. The initial peak of EFS-Induced contraction was tended to be suppresed by ${\alpha},{\beta}$-methylene-adenosine 5'-triphosphate (mATP), a partial agonist of purinergic receptor, and baclofen, a $GABA_B$ receptor agonist (statistically nonsignificant). The late sustained contraction by EFS was suppressed significantly (p < 0.05) by additions of atropione, a cholinergic muscarinic receptor antagonist and baclofen. The adenosine 5'-triphosphate-induced contraction was completely abolished by mA TP but not by baclofen. In the presence of atropine, the subsequent addition of acetylcholine could not contract the muscle strips: but the addition of acetylcholine in the presence of baclofen evoked a contraction to a remarkable extent. These results suggest that in the condition of present study, the cholinergic innervation may play a more important role than the purinergic one, and baclofen suppresses the contractility of rat detrusor by the stimulation of the $GABA_B$ receptors to inhibit the release of neurotransmitter from the cholinergic nerve ending.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.31-36
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• 2011

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express $PSY_1$, $PSY_6$, and $PSY_{11}$ receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to $NAD^+$, an agonist of the human $PSY_{11}$ receptor, and $NADP^+$ as well as ATP, an agonist for $PSY_1$ and $PSY_{11}$ receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by $NAD^+$ and $NADP^+$ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. $NAD^+$ and $NADP^+$ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. $NAD^+$- and $NADP^+$-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.3
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• pp.231-241
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• 2001

The mammalian cortical collecting duct (CCD) plays a major role in regulating renal NaCl reabsorption, which is important in $Na^+$ and $Cl^-$ homeostasis. The M-1 cell line, derived from the mouse cortical collecting duct, has been used as a mammalian model of the study on the electrolytes transport in CCD. M-1 cells were grown on collagen-coated permeable support and short circuit current $(I_{sc})$ was measured. M-1 cells developed amiloride-sensitive current $5{\sim}7$ days after seeding. Apical and basolateral addition of ATP induced increase in $I_{sc}$ in M-1 cells, which was partly retained in $Na^+-free$ or $Cl^--free$ solution, indicating that ATP increased $Na^+$ absorption and $Cl^-$ secretion in M-1 cells. $Cl^-$ secretion was mediated by the activation of apical cystic fibrosis transmembrane regulator (CFTR) chloride channels and $Ca^{2+}-activated$ chloride channels, but $Na^+$ absorption was not mediated by activation of epithelal sodium channel (ENaC). ATP increased cAMP content in M-1 cells. The RT-PCR analysis demonstrated that M-1 cells express $P2Y_2,\;P2X_3\;and\;P2Y_4$ receptors. These results showed that ATP regulates $Na^+$ and $Cl^-$ transports via multiple P2 purinoceptors on the apical and basolateral membranes in M-1 cells.

• Genomics & Informatics
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• v.11 no.4
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• pp.282-288
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• 2013

The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.61-66
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• 2011

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used $Ca^{2+}$ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP ($10\;{\mu}M$) elicited strong but transient $[Ca^{2+}]_i$ increase in a concentration dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking $[Ca^{2+}]_i$ transients were 2MeS-ATP>ATP>>UTP=${\alpha}{\beta}$-MeATP, which was compatible with the subclass of $P2Y_1$ receptor. The $[Ca^{2+}]_i$ transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of $P2Y_1$ selective blocker (MRS 2179; $30\;{\mu}M$). $P2Y_1$ receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, $P2Y_1$ receptor is mainly expressed in a retinoblastoma cell, which elicits $Ca^{2+}$ release from internal $Ca^{2+}$ storage sites via the phospholipase C-mediated pathway. $P2Y_1$ receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic $[Ca^{2+}]_i$ signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.211-219
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• 2004

The effects of adenosine 5'-triphosphate (ATP) and ATP analogs, P/sub 2y/ purinoceptor agonists, on growth of normal mouse mammary epithelial cells (NMuMG) were examined. Cells were plated onto 24 well plates in DMEM supplemented with 10 % fetal calf serum. After serum starvation for 24 hours, ATP, P/sub 2y/ purinoceptor agonists (AdoPP[NH]P, ATP-α-S, ATP-γ-S, β, γ-me-ATP and 2me-S-ATP), P/sub 2u/ purinoceptor agonist (UTP) and P/sub 2y/ purinoceptor antagonists (Reactive Blue 2, more selective to P/sub 2y/ receptor than PPADS; PPADS) were added. DNA synthesis was estimated as incorporation of 3H-thymidine into DNA (1 hour pulse with 1 μ Ci/ml, 18～19 hours after treatment). ATP, Adopp[NH]P, ATP-α-S or ATP-γ-S, significantly increased DNA synthesis at 1, 10 and 100 μM concentrations with dose-dependency (P<0.05), and the maximum responses of ATP and ATP analogs were shown at 100 μM concentration (P<0.05). The potency order of DNA synthesis was ATP≥ATP- γ -S>Adopp [NH]P>ATP-α-S. β, γ -me-ATP, 2me-S-ATP and UTP did not increase DNA synthesis. In autoradiographic analysis of percentage of S-phase cells, similar results were observed to those of DNA synthesis. Addition of 1, 10 or 100 μM Reactive Blue 2 or PPADS significantly decreased ATP (100 μM)-induced DNA synthesis, however, PPADS was less effective than Reactive Blue 2. In Elvax 40P implant experiment, ATP directly stimulated mammary endbud growth in situ suggesting the physiological regulator of ATP in mammary growth. ATP 100 μM rapidly increased MAPK activity, reaching a maximum at 5 min and then gradually decreasing to the base level in 30 min. ATP analogs, Adopp[NH]P and ATP-γ-S also increased MAPK activity, however, β, γ-me-ATP and 2me-S-ATP did not. The inhibitor of the upstream MAPK kinase (MEK), PD 98059 (25 μM), effectively reduced ATP (100 μM) or EGF(10 ng/ml, as positive control)-induced MAPK activity and DNA synthesis (P<0.05). These results indicate that ATP-induced DNA synthesis was prevented from the direct inhibition of MAPK kinase pathway. Overall results support the hypothesis that the stimulatory effects of normal mouse mammary epithelial growth by addition of ATP or ATP analogs are mediated through mammary tissue specific P/sub 2y/ purinoceptor subtype, and MAPK activation is necessary for the ATP-induced cell growth.

• International Journal of Oral Biology
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• v.31 no.4
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• pp.141-148
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• 2006

The effects of adenosine triphosphate(ATP) on salivary glands have been recognized since 1982. The presence of purinergic recepetors(P2Rs) that mediate the effects of ATP in various tissues, including parotid and submandibular salivary gland, has been supported by the cloning of receptor cDNAs and the expression of the receptor proteins. P2Rs have many subtypes, and the activation of these receptor subtypes increase intracellular $Ca^{2+}$, a key ion in the regulation of the secretion in the salivary gland. The apical pores of taste buds in circumvallate and foliate papillae are surrounded by the saliva from von Ebner salivary gland(vEG). Thus, it is important how the secretion of vEG is controlled. This study was designed to elucidate the roles of P2Rs on salivary secretion of vEG. Male Sprague-Dawley rats (about 200 g) were used for this experiment. vEG-rich tissues were obtained from dissecting $500-1,000\;{\mu}m$ thick posterior tongue slices under stereomicroscope view. P2Rs mRNA in vEG acinar cells were identified with RT-PCR. To observe the change in intracellular $Ca^{2+}$ activity, we employed $Ca^{2+}-ion$ specific fluorescence analysis with fura-2. Single acinar cells and cell clusters were isolated by a sequential trypsin/collagenase treatment and were loaded with $10\;{\mu}M$ fura -2 AM for 60 minutes at room temperature. Several agonists and antagonists were used to test a receptor specificity. RT-PCR revealed that the mRNAs of $P2X_4$, $P2Y_1$, $P2Y_2$ and $P2Y_3$ are expressed in vEG acinar cells. The intracellular calcium activity was increased in response to $10\;{\mu}M$ ATP, a P2Rs agonist, and 2-MeSATP, a $P2Y_1$ and $P2Y_2R$ agonist. However, $300\;{\mu}M\;{\alpha}{\beta}-MeATP$, a $P2X_1$ and $P2X_3R$ agonist, did not elicit the response. The responses elicited by $10\;{\mu}M$ ATP and UTP, a $P2Y_2R$ agonists, were maintained when extracellular calcium was removed. $10\;{\mu}M$ suramin, a P2XR antagonist, and reactive blue 2, a P2YR antagonist, partially blocked ATP-induced response. However, when extracellular calciums were removed, suramin did not abolish the responses elicited by ATP. These results suggest that P2Rs play an important role in salivary secretion of vEG acinar cells and the effects of ATP on vEG salivary secretion may be mediated by $P2X_4$, $P2Y_1$, $P2Y_2$, and/or $P2Y_3$.

• The Korean Journal of Pain
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• v.22 no.1
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• pp.1-15
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• 2009

Neuropathic pain is often refractory to intervention because of the complex etiology and an incomplete understanding of the mechanisms behind this type of pain. Glial cells, specifically microglia and astrocytes, are powerful modulators of pain and new targets of drug development for neuropathic pain. Glial activation could be the driving force behind chronic pain, maintaining the noxious signal transmission even after the original injury has healed. Glia express chemokine, purinergic, toll-like, glutaminergic and other receptors that enable them to respond to neural signals, and they can modulate neuronal synaptic function and neuronal excitability. Nerve injury upregulates multiple receptors in spinal microglia and astrocytes. Microglia influence neuronal communication by producing inflammatory products at the synapse, as do astrocytes because they completely encapsulate synapses and are in close contact with neuronal somas through gap junctions. Glia are the main source of inflammatory mediators in the central nervous system. New therapeutic strategies for neuropathic pain are emerging such as targeting the glial cells, novel pharmacologic approaches and gene therapy. Drugs targeting microglia and astrocytes, cytokine production, and neural structures including dorsal root ganglion are now under study, as is gene therapy. Isoform-specific inhibition will minimize the side effects produced by blocking all glia with a general inhibitor. Enhancing the anti-inflammatory cytokines could prove more beneficial than administering proinflammatory cytokine antagonists that block glial activation systemically. Research on therapeutic gene transfer to the central nervous system is underway, although obstacles prevent immediate clinical application.