Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway / 中国中药杂志

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1β(IL-1β) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1β and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

Mechanism of Zhenwu Decoction in improving renal inflammatory injury in mice with DN of spleen-kidney Yang deficiency syndrome by regulating ROCK/IKK/NF-κB pathway / 中国中药杂志

To investigate the intervention effect and mechanism of Zhenwu Decoction on diabetic nephropathy(DN) mice of spleen-kidney Yang deficiency syndrome based on the Rho-associated coiled-coil kinase(ROCK)/IκB kinase(IKK)/nuclear factor-κB(NF-κB) pathway. Ninety-five 7-week-old db/db male mice and 25 7-week-old db/m male mice were fed adaptively for one week. The DN model of spleen-kidney Yang deficiency syndrome was induced by Dahuang Decoction combined with hydrocortisone by gavage, and then the model was evaluated. After modeling, they were randomly divided into a model group, high-dose, medium-dose, and low-dose Zhenwu Decoction groups(33.8, 16.9, and 8.45 g·kg~(-1)·d~(-1)), and an irbesartan group(25 mg·kg~(-1)·d~(-1)), with at least 15 animals in each group. The intervention lasted for eight weeks. After the intervention, body weight and food intake were measured. Serum crea-tinine(Scr), blood urea nitrogen(BUN), fasting blood glucose(FBG), urinary albumin(uALb), and urine creatinine(Ucr) were determined. The uALb/Ucr ratio(ACR) and 24 h urinary protein(UTP) were calculated. Renal pathological morphology was evaluated by HE staining and Masson staining. The levels of key molecular proteins in the ROCK/IKK/NF-κB pathway were detected by Western blot. Enzyme-linked immunosorbent assay(ELISA) was used to detect interleukin-1β(IL-1β), interleukin-6(IL-6), interleukin-8(IL-8), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α). Compared with the blank group, the model group showed increased content of BUN, uALb, and SCr, increased values of 24 h UTP and ACR, decreased content of Ucr(P<0.05), enlarged glomeruli, thickened basement membrane, mesangial matrix proliferation, inflammatory cell infiltration, and collagen fiber deposition. The protein expression of ROCK1, ROCK2, IKK, NF-κB, phosphorylated IKK(p-IKK), phosphorylated NF-κB(p-NF-κB), and phosphorylated inhibitor of NF-κB(p-IκB) increased(P<0.05), while the protein expression of inhibitor of NF-κB(IκB) decreased(P<0.05). The levels of inflammatory factors IL-1β, IL-6, IL-8, and TNF-α increased(P<0.05), while the level of IL-10 decreased(P<0.05). Compared with the model group, the groups with drug treatment showed decreased levels of BUN, uALb, SCr, 24 h UTP, and ACR, increased level of Ucr(P<0.05), and improved renal pathological status to varying degrees. The high-and medium-dose Zhenwu Decoction groups and the irbesartan group showed reduced protein expression of ROCK1, ROCK2, IKK, NF-κB, p-IKK, p-NF-κB, and p-IκB in the kidneys(P<0.05), increased protein expression of IκB(P<0.05), decreased levels of serum inflammatory factors IL-1β, IL-6, IL-8, and TNF-α(P<0.05), and increased level of IL-10(P<0.05). Zhenwu Decoction can significantly improve renal function and renal pathological damage in DN mice of spleen-kidney Yang deficiency syndrome, and its specific mechanism may be related to the inhibition of inflammatory response by down-regulating the expression of key molecules in the ROCK/IKK/NF-κB pathway in the kidney.

The different contraction between rat gastric longitudinal and circular smooth muscle induced by extracellular nucleotides / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To test the different contrctile responses of extracellular nucleotides, such as ATP, UTP and nucleotide uridine adenosine tetraphosphate (Up4A) in gastric longitudinal muscle (LM) and circular muscle (CM). Examined the effect of P2X and P2Y receptor antagonists (in this study, we used IP5I and suramin) and cyclooxygenase inhibitor (indomethacin) on Up4A induced contractile responses in LM and CM.</p><p><b>METHODS</b>The rats were sacrificed and the stomachs were opened to gain LM and CM. Using organ bath system to assess contrctile responses of smooth muscle.</p><p><b>RESULTS</b>Up4A could induce contractile responses in both CM and LM, which were similar with ATP and UTP. IP5 did not attenuate Up4A could induce contractions in both LM and CM, but suramin and indomethacin significantly inhibited Up4A contraction in CM, but not in LM.</p><p><b>CONCLUSION</b>Our results suggest that extracellular nucleosides and their inhibitors induce different responses between LM and CM.</p>

Uridine-5'-Triphosphate Stimulates Chloride Secretion via Cystic Fibrosis Transmembrane Conductance Regulator and Ca2+-Activated Chloride Channels in Cultured Human Middle Ear Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Nucleotide binding to purinergic P2Y receptors contributes to the regulation of fluid and ion transport in the middle ear epithelial cells. Here, we investigated the regulatory mechanism of the P2Y2 receptor agonist, uridine-5'-triphosphate (UTP), on Cl- transport in cultured normal human middle ear epithelial (NHMEE) cells. MATERIALS AND METHOD: Electrophysiological measurements were performed in monolayers of cultured NHMEE cells. Short circuit currents (Isc) were measured from the cells mounted in Ussing chambers under various conditions. RESULTS: Apical addition of UTP in presence of amiloride evoked a transient rise and a sustained response in Isc due to Cl- efflux. Application of different Cl- channel blockers to the apical side of the cells significantly decreased UTP-induced Isc. Niflumic acid (NFA), a known blocker of Ca(2+)-activated chloride channels (CACC), and CFTRinh172, a selective inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR), partially inhibited the UTP-induced Cl- secretion, respectively. CONCLUSION: Cl- transport across the airway epithelia plays a predominant role in regulating airway hydration. In this study, UTP is shown to increase both CACC and CFTR-dependent Cl- secretion in NHMEE cells, suggesting their role in fluid and ion transport in the middle ear epithelium.

Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study.</p><p><b>METHODS</b>The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization.</p><p><b>RESULTS</b>The plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development.</p><p><b>CONCLUSION</b>The antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.</p>

Pharmacological characteristics of contractile responses regulated by P2Y receptors in circular smooth muscle of the rat gastric body / 药学学报

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.

UTP regulates spontaneous transient outward currents in porcine coronary artery smooth muscle cells through PLC-IP(3) signaling pathway / 生理学报

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.

Treatment with MnTBAP Protects Against Early Nuclear Translocation of Endonuclease G and Reduces Cerebral Infarction after Focal Cerebral Ischemia/Reperfusion in Mice

BACKGROUND: Reactive Oxygen Species (ROS) have been implicated in the pathophysiology of brain injury after ischemia/reperfusion. Recently, it has been reported that endonuclease G (EndoG), a mitochondrial protein, is activated by neuronal excitotoxicity and translocated into nucleus inducing apoptosis. However, it is not elucidated whether ROS are involved in the nuclear translocation of EndoG in focal cerebral ischemia/reperfusion in mice. We investigated whether treatment of manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP) protects against early nuclear translocation of EndoG and reduces cerebral infarction after ischemia/reperfusion in mice METHODS: Adult male mice were subjected to middle cerebral artery occlusion (MCAO) for 60 min, followed by reperfusion. Immunohistochemistry and Western blot analysis for EndoG were performed at various time points after ischemia/reperfusion. Double staining with EndoG and Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TUNEL) was also performed. MnTBAP was used to determine whether the production of ROS could inhibit translocation of EndoG into the nucleus. RESULTS: Western blot analysis and Immunohistochemistry of EndoG showed that nuclear EndoG was detected as early as 4 hrs after reperfusion, and mitochondrial EndoG was significantly reduced at the same time. Double staining with EndoG and TUNEL showed a spatial relationship between EndoG expression and DNA fragmentation. MnTBAP-treated mice showed that the translocation of EndoG was attenuated in comparison with the vehicle- treated mice and decreased infarction volume after ischemia/reperfusion. CONCLUSIONS: MnTBAP reduced the generation of ROS, and inhibited the early translocation of EndoG, which was followed by the reduction of infarction volume in the ischemic brain after ischemia/reperfusion.

Effect of Caffeine on UTP-induced Ca2+ Mobilization and Mucin Secretion in Human Middle Ear Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Puringeric receptors and their agonists like uridine-5-triphosphate (UTP) and adenosine triphosphate (ATP), regulate mucin secretion in middle ear epithelial cells. In the present study, we examined the effects of purinergic agonists on Ca2+ influx ([Ca2+]i ) in normal human middle ear epithelial (NHMEE) cells. We also examined the effect of caffeine, an inositol 1, 4, 5-triphosphate (IP3) inhibitor, on UTP induced [Ca2+]i and mucin secretion in NHMEE cells. MATERIALS AND METHOD: NHMEE cells were stimulated with various purinergic agonists, such as UTP, and [Ca2+]i was measured using a miniature double perfusion chamber. UTP-induced mucin secretion was quantitated by immunoblotting assay. RESULTS: The determined order of purinergic agonist potency with respect to [Ca2+]i was ATP=UTP>2-MeSATP>ADP>> adenosine. UTP-induced mucin secretion was inhibited when the intracellular Ca2+ was removed with 2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid-acetoxymethyl ester. Caffeine suppressed UTP-induced [Ca2+]i, and but inhibited UTPinduced and constitutional mucin secretion. CONCLUSION: Our results suggest that caffeine may have a therapeutic effect in mucoid otitis media by suppressing mucin secretion.

Membrane-Specific Expression of Functional Purinergic Receptors in Normal Human Nasal Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Extracellular purines and pyrimidines regulate various physiological responses via cell surface receptors known as purinoreceptors, and may exert autocrine or paracrine effects on ion transport, fluid transport, ciliary beat frequency and mucin secretion. This study aims to investigate the expression patterns of such purinoreceptors found in normal human nasal epithelial (NHNE) cells. MATERIALS AND METHOD: In RT-PCR, the mRNAs for several P2X (P2X3, P2X4, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12) receptors were identified in NHNE cells. Functional localizations of P2 receptors were investigated by measuring [Ca2+]i increases in a membrane-specific manner using a double-perfusion chamber. Absence of the responses of -Me ATP and 2MeS-ATP excluded functionally active P2X3, P2X4, and P2Y1 receptors as far as [Ca2+]i increase was concerned. RESULTS: Applications with ATP and UTP revealed that luminal membranes of NHNE cells express P2Y2 and P2Y6 receptors and basolateral membranes P2Y2 receptors. Expressions of P2Y2 and P2Y6 receptors in NHNE cells were further verified by the immunoblotting using specific antibodies. In addition, the results with BzATP indicated that the P2Y11 receptor may be present on the luminal side. CONCLUSION: The NHNE cells express functionally active P2Y2, P2Y6 and P2Y11 receptors in a membrane-specific pattern, which may play an important role in the control of mucin and fluid secretion in NHNE cells.

The Relationship between the Histological Findings and beta ig-h3 in IgA Nephropathy / 대한신장학회잡지

BACKGROUND: TGF-beta is involved in the pathogenesis of various kidney diseases characterized by glomerulosclerosis and tubulointerstitial fibrosis. It is reported that urinary TGF-beta reflects the grade of interstitial fibrosis in glomerular disease. Here, we evaluated the relationship between the histological findings and beta ig-h3 in IgA nephropathy. METHODS: In patients with IgA nephropathy, we measured blood pressure (BP), serum creatinine, 24-hour urinary protein excretion (UTp), creatinine clearance (Ccr), serum and urine beta ig-h3 levels, and urine TGF-beta levels at the time of renal biopsy. Histologic findings were semiquantitively scored according to the extent of glomerulosclerosis (GG), tubulointerstitial fibrosis (TIG) and hyaline arteriolosclerosis (HA) by the criteria suggested by To. Semiquantitive scoring of immunohistochemistry for beta ig-h3 was done. RESULTS: Mean BP 95.4+/-14.5 mmHg, serum creatinine 1.06+/-0.35 mg/dL, 24-hour UTp 1, 423+/-1, 439 mg/day, and Ccr was 97.84+/-59.73 mL/min. The number of patients that showed GG 3 were 5, GG 2 was 1, GG 1 were 12. And, the number of patients that showed TIG 3 were 2, TIG 2 were 5, TIG 1 were 11. HA was shown in 4 patients. beta ig-h3 immunostaining was observed in glomerular Bowman's capsules and basement membrane of proximal tubules. The degree of beta ig-h3 immunostaining was positively correlated with the degree of glomerulosclerosis (r=0.72, p<0.001), interstitial fibrosis (r=0.91, p<0.001), serum creatinine (r=0.592, p<0.05) and Ccr (r=-0.626, p<0.05), but not with 24-hour UTp. Serum and urine beta ig-h3 levels did not correlate with any of these parameters. CONCLUSION: Renal beta ig-h3 expression in patients with IgA nephropathy may be related to glomerulosclerosis and interstitial fibrosis. However, urinary beta ig-h3 levels did not represent the pathologic changes of IgA nephropathy. Long-term study to measure renal beta ig-h3 expression and urinary beta ig-h3 is required to elucidate the roles of beta ig-h3 in IgA nephropathy.

The Role of ATP on Reactive Oxygen Species Generation and Cellular Proliferation in BV-2 Cells / 대한소아신경학회지

PURPOSE: Adenosine triphosphate (ATP) evokes several cellular responses in microglia including propagation. However, the role of the purinoceptor on ROS generation in microglia is unclear. In order to determine the action of the purinoceptor in microglia, the effects of ATP on ROS generation and cellular proliferation in BV-2 murine microglial cells were evaluated. An additional aim of this study was to investigate signal transduction pathways using several inhibitors. METHODS: The [Ca2+] was measured using Ca2+ sensitive indicator, Fura-2/AM. ROS production was observed by fluorescence-confocal microscope and cell proliferation was evaluated by counting cell number. RESULTS: ATP increased the intracellular calcium levels ([Ca2+]i) in BV-2 cells in a dose-dependent manner. This increase was attenuated by pretreatment with a calcium chelator (EGTA) and a phospholipase C (PLC) inhibitor (U-73122) while the protein tyrosine kinase (PTK) inhibitor (genistein) had no inhibitory effects. To identify the effects of the nucleotides, ROS generation was observed in the nucleotide-stimulated BV-2 cells. The treatment with 100 M ATP induced ROS generation, but 100 M adenosine and 100 M UTP did not. To investigate the signal transduction pathway in ATP-induced ROS generation, several inhibitors were pretreated before adding ATP. ATP- induced ROS production was blocked by pretreatment with either 0.5 mM EGTA or 10 M U73122 while 40 M genistein had an inhibitory effect on ATP action. Correspondingly, 40 M KN62 (CaM kinase II inhibitor), 1 M sphingosine (protein kinase C inhibitor), 1 nM DPI (NADPH oxidase inhibitor) and 50 M mepacrine (phospholipase A2 inhibitor) could suppress ATP-induced ROS generation. The effects of ATP on cell proliferation was observed 3 days after ATP treatment and its peak velocity after 4 days. NF-kB activation was observed after the cells were incubated with 0.1 mM ATP. The maximal level of NF-kB activation was obtained with 0.3 mM ATP while higher concentrations were less effective. CONCLUSION: Overall, we conclude that ATP in BV-2 cells induces ROS generation and cell propagation. The signal transduction pathways including calcium, CaM kinase II, PLC, protein kinase C, phospholipase A2 and NADPH oxidase are involved in ATP-induced ROS generation.

Effect of Uridine 5'-Triphosphate on Mucin Secretion in Human Middle Ear Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Extracellular uridine 5'-triphosphate (UTP) regulates a variety of biological functions in the airway epithelium including chloride and fluid transport, mucociliary clearance, and mucin secretion via P2Y purinergic receptors. This study was undertaken to investigate which P2Y purinergic receptors are expressed in the normal human middle ear epithelial (NHMEE) cells. We also determined the levels of mucin secretion and its mRNA expressions following stimulation with UTP. MATERIALS AND METHOD: The level of P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and P2Y12) receptors and mucin gene 5AC (MUC5AC), MUC5B, MUC8 messenger RNA (mRNA)s were measured by reverse transcription (RT)-polymerase chain reaction (PCR). We also determined the levels of mucin secretion following stimulation with UTP by dot-blotting method in NHMEE cells. RESULTS: Middle ear epithelial cells expressed P2Y1, P2Y2, P2Y6, P2Y11 and P2Y12 receptors but not the P2Y4 receptor. Apically applied UTP induced increased the mucin secretion. On the other hand, UTP did not enhance the mucin mRNA expression until 72 h had lapsed after treatment. CONCLUSION: Our study suggests that UTP acts as a secretogogue on mucin secretion in NHMEE cells.

Validity and safety of sputum induction by HS and UTP in asthma patients

In this study, sputum induction was done on 10 healthy subjects and 24 asthma patients. Sputum induced by inhalation of either hypertonic saline [HS] or uridine 5-triphosphate [UTP] was induced in all the studied subjects. Inhaled UTP produced a larger amount of sputum than HS. There were significant differences in oxygen desaturation during the procedure between the two methods and no significant difference in spirometric measurements during the procedure by both methods. Sputum total cell and differential cell counts with a high proportion of eosinophils in asthmatics were similar between specimens obtained by HS and UTP. Therefore, sputum induction either by HS or UTP is noninvasive, relatively safe, reproducible and repeatable measurement. Patients with asthma have a higher proportion of eosinophils in their induced sputum with a significant negative correlation to FEV1% pred. The analysis of induced sputum has a relevant role in the identification and monitoring of airway inflammation

Probing the nucleotide binding sites in T7 RNA polymerase using cibacron blue.

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. In this work we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, and its effect on the function of the enzyme. T7 RNAP binds to the dye in a bi-phasic manner. The first phase of the binding is characterized by a high affinity (Kd in the nanomolar range) and reversible inactivation of the enzyme. The second binding site is the common substrate binding site. The association of the dye with T7 RNAP is a good model to understand the physiological significance of a high affinity binding of the initiating nucleotide, GTP, earlier reported from our laboratory. The results will be discussed to understand the role of the high affinity GTP binding.

Asymmetrical distribution of P2Y nucleotide receptors in rabbit inner medullary collecting duct cells

We cultured the rabbit inner medullary collecting duct (IMCD) cells as monolayers on collagen-coated membrane filters, and investigated distribution of the P2Y receptors by analyzing nucleotide-induced short circuit current (Isc) responses. Exposure to different nucleotides of either the apical or basolateral surface of cell monolayers stimulated Isc. Dose-response relationship and cross-desensitization studies suggested that at least 3 distinct P2Y receptors are expressed asymmetrically on the apical and basolateral membranes. A P2Y2-like receptor, which responds to UTP and ATP, is expressed on both the apical and basolateral membranes. In addition, a uracil nucleotide receptor, which responds to UDP and UTP, but not ATP, is expressed predominantly on the apical membrane. In contrast, a P2Y1-like receptor, which responds to ADP and 2-methylthio-ATP, is expressed predominantly on the basolateral membrane. These nucleotides stimulated intracellular cAMP production with an asymmetrical profile, which was comparable to that in the stimulation of Isc. Our results suggest that the adenine and uracil nucleotides can interact with different P2Y nucleotide receptors that are expressed asymmetrically on the apical and basolateral membranes of the rabbit IMCD cells, and that both cAMP- and Ca2+-dependent signaling mechanisms underlie the stimulation of Isc.

Vascular Reactivity by Purinoceptor Activation in Rat Inferior Vena Cava

BACKGROUND: Extracellular ATP, released from platelets and nerve endings, plays significant roles in the regulation of circulation. The effects of ATP depend on the location of the vessels and the species of experimental animals. Until now, studies were limited to arteries, so we compared the effects of ATP in rat vena cava with those in the aorta and attempted to identify the characteristics of their receptors. METHODS: Vascular rings were isolated from the rat inferior vena cava and descending thoracic aorta. Endothelial cells were preserved or removed by gentle rubbing. The isometric contractions were recorded on polygraph using a force transducer. RESULTS: In the vena cava ring precontracted by 100 nM norepinephrine (NE), ATP elicited relaxations in a dose-dependent manner. These effects were abolished by removal of the endothelium or pretreatment with a nitric oxide synthase inhibitor. Relaxations to ATP in the vena cava (EC50 :9.9 microM) were less potent than those in the aorta (1.7 microM). The relative order of potencies was ADP>ATP>AMP>adenosine, but the maximal relaxation to ADP was smaller than to ATP. ATP-induced vasorelaxation was blocked by suramin, a nonselective antagonist for P2 purinoceptor and reactive blue-2, a P2Y blocker. At basal tension, ATP contracted the vena cava dose-dependently and these effects were potentiated by endothelium-removal. Contractions in the vena cava were also less potent than in the aorta, and the order of potencies was alpha, beta-MeATP>UTP>ATP>ADP>AMP=adenosine. ATP-induced vasoconstriction was blocked by suramin and alpha, beta-MeATP, a desensitizing antagonist of P2X purinoceptor, and potentiated by pretreatment with UTP. CONCLUSION: These results suggest that ADP and ATP acts on P2Y1- and P2Y2-purinoceptor in the endothelium, respectively and induces vasorelaxation of the vena cava, which is mediated by nitric oxide. Since ATP and UTP induced vasoconstriction in endothelium-denuded condition, it may be mediated by the activation of the P2X and P2Y4, 6 purinoceptor on smooth muscles, respectively.

Evidence for adenosine triphosphate (ATP) as an excitatory neurotransmitter in guinea-pig gastric antrum

We explore the question of whether adenosine 5'-triphosphate (ATP) acts as an excitatory neurotransmitter in guinea-pig gastric smooth muscle. In an organ bath system, isometric force of the circular smooth muscle of guinea-pig gastric antrum was measured in the presence of atropine and guanethidine. Under electrical field stimulation (EFS) at high frequencies (>20 Hz), NO-mediated relaxation during EFS was followed by a strong contraction after the cessation of EFS (a "rebound-contraction"). Exogenous ATP mimicked the rebound-contraction. A known P2Y-purinoceptor antagonist, reactive blue 2 (RB-2), blocked the rebound-contraction while selective desensitization of P2x-purinoceptor with alpha, beta-MeATP did not affect it. ATP and 2-MeSATP induced smooth muscle contraction, which was effectively blocked by RB-2 and suramin, a nonselective P2-purinoceptor antagonist. Particularly, in the presence of RB-2, exogenous ATP and 2-MeSATP inhibited spontaneous phasic contractions, suggestingthe existence of different populations of purinoceptors. Both the rebound-contraction and the agonist-induced contraction were not inhibited by indomethacin. The rank orders of agonists' potency were 2-MeSATP > ATP gtoreq UTP for contraction and alpha, beta-MeATP gtoreq beta, gamma-MeATP for inhibition of the phasic contraction, that accord with the commonly accepted rank order of the classical P2Y-purinoceptor subtypes. Electrical activities of smooth muscles were only slightly influenced by ATP and 2-MeSATP, whereas alpha, beta-MeATP attenuated slow waves with membrane hyperpolarization. From the above results, it is suggested that ATP acts as an excitatory neurotransmitter, which mediates the rebound-contraction via P2Y-purinoceptor in guinea-pig gastric antrum.

Mechanisms of the UTP-induced tension in mammalian skeletal muscles

The mechanisms of UTP-induced tension in human and rat skinned fibers were investigated using isometric tension recordings, electrophysiological techniques and biochemical methods. In fast-type fibers from rat extensor digitorum longus (EDL) the UTP-induced tension: a) required previous loading of CA2+ into the sarcoplasmic reticulum (SR); b) was inhibited by previous exposure to caffeine; c) was abolished by functional disruption of the SR; d) was not affected by blockade of the SR Ca2+-release channels by ruthenium red or heparin; e) was prevented by spermidine. These data point to the SR as the target of UTP action and suggest a pathway of UTP-induced Ca2+-release independent of the ryanodine- or the IP3-sensitive Ca2+-release channels. Accordingly, UTP failed to stimulate the electrophysiological activity of ryanodine-sensitive channels, incorporated into lipid bilayers. We suggest that UTP-induced Ca2+-release might occur via the channel form of the SR Ca2+-ATPase. The UTP-induced tension in human slow-type fibers was not affected by the SR Ca2+ content or by disruption of the SR, but was accompanied by changes in the tension-pCa relationship, namely increase in maximum Ca2+-activated tension, and in apparent Ca2+-affinity of troponin. The UTP-induced tension in slow-type fibers from rat soleus was partially inhibited by Ca2+-depletion from, or by disruption of the SR, and was accompanied by changes in tension/pCa relationship, similar to those observed in human fibers. Both in skinned fibers and in isolated SR vesicles, UTP was less effective than ATP as a substrate for the SR Ca2+-ATPase. This effect might contribute to UTP-induced tension.

In vitro Transcription of Prospect Hill virus S Genome Segment Produced by RT-PCR

Prospect Hill virus (PHV) is related antigenically but distinct to Hantaan virus. As a member of genus Hantavirus, PHV has three segmented RNA genome. Among these segments, Small segment(S) encodes nucleocapsid protein (NP) as structural protein and also may do functional nonstructural protein(NSs). We performed in vitro transcription to produce vRNA of PHV S genome. For the first step of in vitro transcription of S genome of PHV, the S RNA segrnent which is 1,675 nucleotides long was amplified by RT-PCR using PCR primers built according to cDNA sequence of PHV S genome. Next, a new PCR primer appended above downstream primer to T7 phage promoter sequence was reconstructed to obtain PCR product containing T7 promoter. Then another PCR was performed. Using this PCR product as the template, in vitro run-off transcription without cloning by transcriptional vector was performed to obtain viral- sense RNA transcript. Thereafter, the size of transcript was assessed by running on formaldehyde agarose gels. Since the transcription reactants contain a-S UTP, the transcript is detectable by autoradiography. The transcript was also detectable by northern hybridization with a-P dCTP- labelled PHV amplicon probe (319 bp) and the initiation start point of run-off transcription was also determined by primer extention analysis. Our data indicate that the in vitro transcript could be produced from the PCR product amplified by PCR primer containing T7 phage promoter without cloning into a phage transcription vector.