1.Effect and mechanism of VEGF antisense phosphorothioate deoxynucleotides on HL-60 leukemic cells.
Journal of Experimental Hematology 2004;12(5):640-643
To explore dose-effect or time-effect of vascular endothelial growth factor (VEGF) antisense phosphorothioate oligodeoxynucleotides (AS PS-ODN) on growth of HL-60 cells, and to study the effect mechanism so as to find new role of VEGF, A7, which was the most effective one of AS PS-ODN selected with computer-aided design and experimental assay, contains 20-DNA modified with phosphorothioate and was tranferred into cells mediated with lipofectin. After culture for 72 hours, inhibitive rate of cell growth was detected with MTT methods, viable cells were counted with trypan blue exclusion each 24 hour, cell configuration and apoptosis were observed with Geimsa staining and flow cytometry respectively, level of VEGF protein was detected with VEGF ELISA kit. The results showed that A7 is able to inhibit cell growth of HL-60 in dose-depending manner of AS PS-ODN, to down-regulate VEGF protein expression significantly, and not to induce apoptosis of HL-60 cells. It is concluded that there is possibility that the inhibition effect of VEGF AS PS-ODN on HL-60 cell growth is to restrain cell proliferation without inducing apoptosis of HL-60 cell, which would interpret that endogenous VEGF proteins have a capacity of promoting proliferation of HL-60 cell.
Cell Proliferation
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drug effects
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Flow Cytometry
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HL-60 Cells
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Humans
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Oligonucleotides, Antisense
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pharmacology
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Thionucleotides
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pharmacology
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Vascular Endothelial Growth Factor A
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analysis
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physiology
2.In vitro study of the effects of arsenic trioxide combined with 8-CPT-cAMP on differentiation induction in retinoic acid resistant acute promyelocytic leukemia cells.
Qi ZHU ; Yun YU ; Pei-min JIA ; Xun CAI ; Sai-juan CHEN ; Zhu CHEN ; Zhen-yi WANG ; Jian-hua TONG
Chinese Journal of Hematology 2003;24(1):6-9
OBJECTIVETo investigate the potential effects of arsenic trioxide (As(2)O(3)) combined with 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the retinoic acid (RA)-resistant acute promyelocytic leukemia (APL) cells.
METHODSThe RA resistant APL cell lines NB4-R1 and NB4-R2 were used as in vitro models. The effect of As(2)O(3) and/or 8-CPT-cAMP was evaluated according to cellular morphology, cell surface antigen and nitroblue-tetrazolium (NBT) assay. Meanwhile, immunofluorescence analysis and Western blot assay were used to detect the degradation of PML-RAR alpha fusion protein and the change of several key cell cycle regulatory proteins in these cells before and after the treatment.
RESULTSLow dose of As(2)O(3) (0.25 micromol/L) synergized with 8-CPT-cAMP (200 micromol/L) in inducing differentiation of NB4-R1 and NB4-R2 cells, while neither of these two drugs alone could induce differentiation of these cells. In addition, 8-CPT-cAMP was able to inhibit the cell growth by modulating the expression of some important cell cycle regulators and to facilitate the As(2)O(3)-mediated degradation of PML-RAR alpha fusion protein.
CONCLUSIONSAs(2)O(3) combined with 8-CPT-cAMP could induce differentiation of RA-resistant APL cells.
Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Cyclic AMP ; analogs & derivatives ; pharmacology ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm ; Drug Synergism ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Oxides ; pharmacology ; Thionucleotides ; pharmacology ; Tretinoin ; pharmacology
3.Enhancement of GABA-activated currents by arginine vasopressin in rat dorsal root ganglion neurons.
Fang QIU ; Wang-Ping HU ; Zhi-Fan YANG
Acta Physiologica Sinica 2014;66(6):647-657
A growing number of studies have shown that arginine vasopressin (AVP) plays an analgesia role in the modulation of nociception. Previous studies have focused on the central mechanisms of AVP analgesia. The aim of the present study was to find out whether peripheral mechanisms are also involved. The effect of AVP on GABA-activated currents (IGABA) and GABAA receptor function in freshly isolated dorsal root ganglion (DRG) neurons of rats were studied using whole cell patch clamp technique. The result showed that, IGABA were potentiated by pre-treatment with AVP (1 × 10⁻¹⁰-1 × 10⁻⁵ mol/L) in a concentration-dependent manner. Meanwhile, the GABA concentration-response curve was shifted upwards, with an increase of (49.1 ± 4.0)% in the maximal current response but with no significant change in the EC50 values. These results indicate that the enhancing effect is non-competitive. In addition, the effects of AVP on IGABA might be voltage-independent. This potentiation of IGABA induced by AVP was almost completely blocked by the V1a receptor antagonist SR49059 (3 × 10⁻⁶ mol/L). Also it could be removed by intracellular dialysis of either GDP-β-S (5 × 10⁻⁴mol/L), a non-hydrolyzable GDP analog, or GF109203X (2 × 10⁻⁶ mol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. These results suggest that AVP up-regulates the function of the GABAA receptor via G protein-coupled receptors and PKC-dependent signal pathways in rat DRG neurons, and this potentiation may underlie the analgesia induced by AVP.
Animals
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Arginine Vasopressin
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pharmacology
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Ganglia, Spinal
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cytology
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Guanosine Diphosphate
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analogs & derivatives
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pharmacology
;
Indoles
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Maleimides
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Membrane Potentials
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Neurons
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drug effects
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Patch-Clamp Techniques
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Rats
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Rats, Sprague-Dawley
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Receptors, GABA-A
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metabolism
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Signal Transduction
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Thionucleotides
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pharmacology
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gamma-Aminobutyric Acid
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pharmacology
4.The effect of telomerase inhibitors on oral squamous carcinoma cells.
Zhiqiang YANG ; Wen CHEN ; Qiao ZHANG
West China Journal of Stomatology 2003;21(3):231-234
OBJECTIVETo study the potential anti-tumor effect of telomerase inhibitors.
METHODSHuman oral squamous carcinoma cell line KB was selected as target cell. The effects of 3'-azido-3'-deoxythymidine (AZT) and human antisense phosphorothioate (AS-ONS) for telomerase template on KB cell line were investigated. The cytotoxic effect of AZT and AS-ONS on tumor cells was quantified using MTT colorimetric assay. Assay of 3H-TdR incorporation was undertaken to measure the cell proliferation. The changes of telomerase activity after treatment was detected and quantified by telomeric repeat amplification protocol (TRAP) semi-quantitative analysis. The Flow Cytometry was used to detect apoptosis and measure cell cycle.
RESULTSBoth AZT and AS-ONS inhibited the growth of KB cell line in a certain range of concentrations, and meanwhile the telomerase activity was reduced after treatment. In addition, both AZT and AS-ONS can induce apoptosis and arrest G1 phase of cell cycle.
CONCLUSIONThe results obtained above indicated that AZT and AS-ONS could be potentially used as an anti-oral carcinoma agent or an auxiliary treatment for cancer. Those inhibitory effects might be partially due to the induction of apoptosis and the prolongation of cell cycle.
Antineoplastic Agents ; pharmacology ; Carcinoma, Squamous Cell ; pathology ; Cell Division ; drug effects ; Enzyme Inhibitors ; pharmacology ; Humans ; Mouth Neoplasms ; pathology ; Oligodeoxyribonucleotides ; pharmacology ; Telomerase ; antagonists & inhibitors ; metabolism ; Thionucleotides ; Tumor Cells, Cultured ; Zidovudine ; pharmacology
5.Inhibition of HBV replication by antisense oligodeoxyribonucleotides in HepG2 cells transfected with a cloned HBV DNA.
Sang Hwan OH ; Byung Il YEH ; Seok Hyun KIM
Yonsei Medical Journal 1995;36(6):527-533
The effect of antisense oligodeoxyribonucleotides(oligo[dN]s) on hepatitis B virus(HBV) replication in HepG2 cells harboring a cloned HBV genome was examined. Antisense oligo(dN)s directed at translational initiation sites of S, pre C and P genes of HBV were treated to the cells and the amount of HBsAg and HBV DNA content were measured 72 hours after the treatment. HBsAg expressions in HepG2 cells harboring the HBV genome were inhibited 68%, 53%, and 46% by the treatment with antisense oligo[dN] directed at S, pre C, and P gene loci, respectively, and HBV DNA content in the cells was also reduced by the treatment of each antisense oligo[dN]. The doubling times of the cultured cells treated with 25 micrograms, 50 micrograms, and 100 micrograms of antisense oligo[dN]/ml medium were 43.3, 62.1, and 93.0 hours, respectively, compared with 37.5 hours of the untreated control cells. Cellular DNA synthesis was inhibited by the treatment with 100 micrograms/ml of antisense oligo [dN], however, no significant effect was observed by the treatment with 50 micrograms or less of antisense oligo[dN]/ml. These results suggested that antisense oligo[dN]s specific to the translational initiation sites of S, pre C, and P genes of HBV may have therapeutic potential for the suppression of HBV propagation in chronic HBV infected patients.
Cloning, Molecular
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DNA, Viral/*genetics
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Hepatitis B Virus/genetics/*physiology
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Hepatoblastoma/pathology/*virology
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Human
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Liver Neoplasms/pathology/*virology
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Oligonucleotides, Antisense/*pharmacology
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Support, Non-U.S. Gov't
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Thionucleotides/*pharmacology
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Transfection
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Tumor Cells, Cultured/virology
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*Virus Replication
6.cAMP analogue 8-CPT-cAMP inducing differentiation in the M2b subtype of acute myeloid leukemia cell line Kasumi-1.
Qi ZHU ; Jun-Pei HU ; Pei-Min JIA ; Zhen-Yi WANG ; Jian-Hua TONG
Journal of Experimental Hematology 2008;16(1):44-47
This study was aimed to investigate the possible effects of cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio) adenosine 3', 5'-cyclic monophosphate (8-CPT-cAMP) on the M(2b) subtype of acute myeloid leukemia (AML-M(2b)) cells. AML-M(2b) is characterized by the non-random chromosome translocation t (8; 21) (q22; q22), through which AML1 (acute myeloid leukemia 1) gene on chromosome 21 is fused with ETO (eight twenty-one) gene on chromosome 8, coding correspondent AML1-ETO fusion protein, which plays a crucial role in the leukemogenesis of AML-M(2b). The AML-M(2b) cell line Kasumi-1 cells were used as an in vitro model. The influences of 8-CPT-cAMP on the proliferation and differentiation of Kasumi-1 cells were evaluated according to cellular morphology, changes in cell surface antigen and cell cycle, as well as nitroblue-tetrazolium (NBT) assay. Meanwhile, semi-quantity RT-PCR and Western blot assay were used to detect the degradation of AML1-ETO fusion protein in Kasumi-1 cells before and after the treatment. The results showed that 8-CPT-cAMP (200 micromol/L) could significantly inhibit cell growth and induce differentiation of Kasumi-1 cells. However, it must be pointed out that 8-CPT-cAMP-induced differentiation in Kasumi-1 is not a typical terminal differentiation. Furthermore, 8-CPT-cAMP exerted little influence on the expression of AML1-ETO fusion gene and its product in Kasumi-1 cells. In conclusion, the 8-CPT-cAMP induced differentiation in Kasumi-1 cells. This results may provide experimental and theoretical basis for the breakthrough of differentiation-induced therapy extended to another leukemia.
Cell Transformation, Neoplastic
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drug effects
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Core Binding Factor Alpha 2 Subunit
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genetics
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metabolism
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Cyclic AMP
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analogs & derivatives
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pharmacology
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Humans
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Leukemia, Myeloid, Acute
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genetics
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metabolism
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pathology
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Oncogene Proteins, Fusion
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genetics
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metabolism
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RUNX1 Translocation Partner 1 Protein
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Thionucleotides
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pharmacology
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Tumor Cells, Cultured
7.In vivo inhibition of hepatitis B virus replication and gene expression by targeted phosphorothioate modified antisense oligodeoxynucleotides.
Sen ZHONG ; Su Jun ZHENG ; Feng CHEN ; Shou Ming WEN ; Sheng Qi WANG ; Jian Jun ZHANG ; Chun Liang DENG
Chinese Journal of Hepatology 2002;10(4):283-286
OBJECTIVETo investigate the antiviral effect of targeted antisense oligodeoxynucleotides (asODN) in HBV transgenic mice.
METHODSasODN phosphorothioated (5'-CATGCCCCAAAGCCAC-3') targeted to HBV pre-C/C region was synthesized. Gal15-PLL was used as drugs carrier which targeted asODN to mice liver. Twelve mice with positive serum HBsAg, HBV-DNA were divided into the Gal15-PLL-asODN-treated group or the control group randomly. In Gal15-PLL- asODN-treated group, each mouse was injected i.v. asODN 15mug/g weighty/day via tail vein for 12 days successively; while in the control group, each mouse received the same volume normal saline by the same way.
RESULTSIn the Gal15-PLL- asODN-treated group, serum HBsAg decreased at the 6th day (P<0.05), and decreased significantly at the 12th day vs pretreatment (P<0.01). The serum HBV DNA of 4/6 mice became negative. Immunohistochemistry test showed lowered HBsAg, HBcAg content in the liver. In contrast, the control group showed no apparent changes.
CONCLUSIONSGal15-PLL-asODN targeted to pre-C/C region could inhibit HBV replication and gene expression.
Animals ; DNA, Viral ; blood ; Gene Expression ; drug effects ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Mice ; Mice, Transgenic ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Thionucleotides ; pharmacology ; Virus Replication ; drug effects
8.Effects of purinergic analogues on spontaneous contraction and electrical activities of gastric antral circular muscle in guinea-pig.
Zheng JIN ; Hui-Shu GUO ; Dong-Yuan XU ; Ming-Yu HONG ; Xiang-Lan LI ; Wen-Xie XU
Acta Physiologica Sinica 2004;56(6):678-684
In order to investigate the role of non-adrenergic non-cholinergic nerves in regulating mechanical and electrical activity of gastric circular smooth muscle, the effects of ATP and its analogues on gastric motility and electrical activities were observed in guinea-pig. In organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured. Electrical activity of the muscle was recorded by using intracellular microelectrode. Electrical and mechanical activities were recorded by chart recorder. ATP and 2-MeSATP potentiated the mechanical activity but did not affect electrical activity in gastric circular smooth muscle. ATP and 2-MeSATP-induced contraction was effectively blocked by nonselective P2y-purinoceptor antagonist, reactive-blue-2 and suramin, but ATP-induced contraction was not blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. However, alpha,beta-MeATP, P2x-purinoceptor agonist, attenuated slow waves with membrane hyperpolarization and inhibited contraction. The relaxation by beta,gamma-MeATP was blocked by alpha,beta-MeATP-induced desensitization of P2x-purinoceptors. ATP-induced contraction was blocked by external calcium-free, but not by nicardipine, a L-type calcium channel blocker. Indomethacin, a nonselective cyclooxygenase inhibitor, did not block ATP-induced contraction. The results suggest that: (1) ATP- and analogues-induced contraction is mediated by P2y-purinoceptor, whereas alpha,beta-MeATP-induced relaxation by P2x-purinoceptor in guinea-pig gastric antral circular smooth muscle. (2) ATP-induced contraction is dependent on extracellular calcium, but Ca2+ entry is not mediated by L-type calcium channel. (3) Prostaglandins are not involved in ATP- and analogue-induced contraction of gastric circular smooth muscle in guinea-pigs, and alpha,beta-MeATP-induced relaxation is related to membrane hyperpolarization.
Adenosine Triphosphate
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analogs & derivatives
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pharmacology
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Animals
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Electrophysiology
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Guinea Pigs
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In Vitro Techniques
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Microelectrodes
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Muscle Contraction
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drug effects
;
physiology
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Muscle, Smooth
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drug effects
;
physiology
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Purinergic Agonists
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Pyloric Antrum
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drug effects
;
physiology
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Thionucleotides
;
pharmacology
9.Pharmacological characteristics of contractile responses regulated by P2Y receptors in circular smooth muscle of the rat gastric body.
Acta Pharmaceutica Sinica 2009;44(5):473-479
This study is to observe the difference in pharmacological characteristics between circular smooth muscles of rat isolated gastric body and gastric fundus, and to investigate the effects of nucleoside and nucleotide on circular smooth muscle of the rat gastric body and the involved receptors. Circular muscle strips of the rat gastric body and gastric fundus were prepared, and contractile responses to agonists were investigated with a technique of drug-receptor interaction in functional system. There was no significant difference between the circular muscle strips of the gastric body and gastric fundus in the responses to KCl, and no difference in EC50 values of contractile responses for 5-HT and His between the two kinds of preparations (P > 0.05). However, Emax values of contractile responses to 5-HT and His [(0.81 +/- 0.26) and (0.88 +/- 0.27) g] in gastric body were significantly smaller than those in gastric fundus [(2.67 +/- 0.61) and (1.90 +/- 0.68) g, P < 0.01], and EC50 value of CCh produced contractile response [(0.45 +/- 0.15) micromol x L(-1)] in gastric body was significantly higher than that in gastric fundus [(0.20 +/- 0.09) micromol x L(-1), P < 0.01]. In precontracted circular muscle strips of the gastric body, ATP (0.1-3000 micromol x L(-1)) produced only a contractile response concentration-dependently, but the same concentration of ATP induced a biphasic response (relaxation followed by a contraction) in precontracted circular muscle strips of the gastric fundus. ATP, UTP, ADP, 2-MeSATP and alpha,beta-MeATP produced contractile responses concentration-dependently in circular muscle strips of the rat gastric body. The EC50 value for 2-MeSATP [(7.2 +/- 5.2) nmol x L(-1)] was about 500 times lower than that for Ach [(3.47 +/- 1.20) micromol x L(-1)]. The rank order of potency for the contraction was 2-MeSATP>ADP>ATP=UTP>alpha,beta-MeATP>adenosine. The contractile responses to ATP and UTP were not significantly affected by phentolamine, propranolol, atropine or tetrodotoxin. In conclusion, there is a significant difference in pharmacological characteristics between the circular smooth muscles of the rat gastric body and gastric fundus and nucleotides might be important mediators responsible for the contraction via a specific P2Y receptor in circular smooth muscle of the rat gastric body.
Adenosine
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pharmacology
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Adenosine Diphosphate
;
pharmacology
;
Adenosine Triphosphate
;
analogs & derivatives
;
pharmacology
;
Animals
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Gastric Fundus
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drug effects
;
physiology
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Male
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Muscle Contraction
;
drug effects
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Muscle, Smooth
;
drug effects
;
physiology
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Purinergic P2 Receptor Agonists
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Rats
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Rats, Wistar
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Receptors, Purinergic P2
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drug effects
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Stomach
;
drug effects
;
physiology
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Thionucleotides
;
pharmacology
;
Uridine Triphosphate
;
pharmacology
10.Scutellarin attenuates endothelium-dependent aasodilation impairment induced by hypoxia reoxygenation, through regulating the PKG signaling pathway in rat coronary artery.
Ya-Juan CHEN ; Lei WANG ; Guang-Yu ZHOU ; Xian-Lun YU ; Yong-Hui ZHANG ; Na HU ; Qing-Qing LI ; Chen CHEN ; Chen QING ; Ying-Ting LIU ; Wei-Min YANG
Chinese Journal of Natural Medicines (English Ed.) 2015;13(4):264-273
Scutellarin (SCU), a flavonoid from a traditional Chinese medicinal plant. Our previous study has demonstrated that SCU relaxes mouse aortic arteries mainly in an endothelium-depend-ent manner. In the present study, we investigated the vasoprotective effects of SCU against HR-induced endothelial dysfunction (ED) in isolated rat CA and the possible mechanisms involving cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG). The isolated endothelium-intact and endothelium-denuded rat CA rings were treated with HR injury. Evaluation of endothelium-dependent and -independent vasodilation relaxation of the CA rings were performed using wire myography and the protein expressions were assayed by Western blotting. SCU (10-1 000 μmol·L(-1)) could relax the endothelium-intact CA rings but not endothelium-denuded ones. In the intact CA rings, the PKG inhibitor, Rp-8-Br-cGMPS (PKGI-rp, 4 μmol·L(-1)), significantly blocked SCU (10-1 000 μmol·L(-1))-induced relaxation. The NO synthase (NOS) inhibitor, NO-nitro-L-arginine methylester (L-NAME, 100 μmol·L(-1)), did not significantly change the effects of SCU (10-1 000 μmol·L(-1)). HR treatment significantly impaired ACh-induced relaxation, which was reversed by pre-incubation with SCU (500 μmol·L(-1)), while HR treatment did not altered NTG-induced vasodilation. PKGI-rp (4 μmol·L(-1)) blocked the protective effects of SCU in HR-treated CA rings. Additionally, HR treatment reduced phosphorylated vasodilator-stimulated phosphoprotein (p-VASP, phosphorylated product of PKG), which was reversed by SCU pre-incubation, suggesting that SCU activated PKG phosphorylation against HR injury. SCU induces CA vasodilation in an endothelium-dependent manner to and repairs HR-induced impairment via activation of PKG signaling pathway.
Animals
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Apigenin
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pharmacology
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Cell Adhesion Molecules
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drug effects
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Cell Hypoxia
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Coronary Vessels
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drug effects
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Cyclic GMP
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analogs & derivatives
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metabolism
;
pharmacology
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Cyclic GMP-Dependent Protein Kinases
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Glucuronates
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pharmacology
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Microfilament Proteins
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drug effects
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NG-Nitroarginine Methyl Ester
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metabolism
;
pharmacology
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Phosphoproteins
;
drug effects
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Rats
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Rats, Sprague-Dawley
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Reperfusion Injury
;
complications
;
physiopathology
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Signal Transduction
;
drug effects
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Thionucleotides
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metabolism
;
pharmacology
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Vasodilation
;
drug effects
;
physiology