Biomedical subjects

Takeshi Katsuragi

Publications and source records attributed to Takeshi Katsuragi.

At least 19 recordsLinked to original sources

PubMed2006

A novel method for preserving human lungs using a super-cooling system.

PURPOSE: To ensure the suitable preservation of isolated lungs, a super-cooling system was used to cool water to temperatures as low as -5 degrees C without freezing. DESCRIPTION: After lung tissues were obtained from patients with lung cancer, they were kept at -5 degrees C or 4 degrees C for as many as 5 days, and then they were histologically and biochemically examined. To evaluate biochemical stability, tissues after storage were passively sensitized with immunoglobulin E and then incubated with anti-immunoglobulin-E antibody. EVALUATION: Although tissues preserved at -5 degrees C for 5 days had an almost normal appearance with intact cilia on bronchial epithelium and normal endothelium, tissues stored at 4 degrees C showed degradation of these structures. Single-stranded DNA, a sign of DNA cleavage, was frequently noted in tissues stored at 4 degrees C, but only rarely observed at -5 degrees C. A significant amount of cysteinyl-leukotrienes was generated from tissues stored at -5 degrees C for 3 days, but there was no response to antibody stimulation from tissues stored at 4 degrees C. CONCLUSIONS: Super-cooling systems may provide useful applications as a novel preserving method.

Aged↗

PubMed2005

Effects of inducible nitric oxide synthase inhibitors on asthma depending on administration schedule.

The effectiveness of two inducible nitric oxide synthase (iNOS) inhibitors on allergic airway inflammation was investigated under different administration schedules. Rats sensitized to ovalbumin (OVA) were exposed to OVA for 3 consecutive days. Both iNOS inhibitors showed markedly different effects between two pretreatment schedules: pretreatment before each of three OVA exposures S1 and before the third exposure alone S2. S1 pretreatment resulted in higher pulmonary resistance than triple OVA alone. This potentiation was associated with increased eosinophil infiltration and malondialdehyde levels in the lungs, which were suppressed by superoxide dismutases (SODs) but not by methylprednisolone. However, the S2 administration of both iNOS inhibitors completely suppressed the airway response. Administration by schedule S1 completely suppressed plasma nitrite and nitrate levels, but that by S2 caused only a slight suppression. The triple OVA exposures resulted in the upregulation of iNOS in alveolar macrophages and arginase activity, Mn- and Cu/Zn-SOD expression, and nitrotyrosine and lipid peroxide deposition in the airway. However, inhibitors administered by schedule S1 suppressed this upregulation, but further potentiated nitrotyrosine, which in turn was inhibited by SOD. Although iNOS inhibitors may be beneficial for asthma, repeated administration may be detrimental because of extensive reduction of NO and downregulation of SOD.

Amidines↗

PubMed2005

A new approach to finding specific dopamine D4 receptor agonists.

This paper describes a new approach to finding specific dopamine D4 receptor agonists based on pharmacological analysis of the contractile response to ATP in guinea pig vas deferens. A partial cDNA of the dopamine D4 receptor of the vas deferens was identified. In the vas deferens, reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis revealed the existence of dopamine D4 receptor mRNA and D4 receptor protein, respectively. ATP (10(-7) M) induced a transient phasic contraction in the presence of prazosin (10(-7) M), an alpha1-adrenoceptor antagonist. This contraction was potentiated by dopamine receptor agonists in a concentration-dependent manner; and was antagonized by 8-Methyl-6-(4-methyl-1-piperazinyl)-11H-pyrido[2,3-b][1,4]benzodiazepine (JL-18), a dopamine D4 receptor antagonist, but not by raclopride, a dopamine D2 and D3 receptor antagonist. Assay methods utilizing contractile responses to ATP may be available for identifying novel dopamine D4 receptor agonists.

Adenosine Triphosphate↗

PubMed2005

Adenosine induces ATP release via an inositol 1,4,5-trisphosphate signaling pathway in MDCK cells.

ATP is released into extracellular space as an autocrine/paracrine molecule by mechanical stress and pharmacological-receptor activation. Released ATP is partly metabolized by ectoenzymes to adenosine. In the present study, we found that adenosine causes ATP release in Madin-Darby canine kidney cells. This release was completely inhibited by CPT (an A1 receptor antagonist), U-73122 (a phospholipase C inhibitor), 2-APB (an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) receptor blocker), thapsigargin (a Ca2+-ATPase inhibitor), and BAPTA/AM (an intracellular Ca2+ chelator), but not by DMPX (an A2 receptor antagonist). However, forskolin, epinephrine, and isoproterenol, inducers of cAMP accumulation, failed to release ATP. Adenosine increased intracellular Ca2+ concentrations that were strongly blocked by CPT, U-73122, 2-APB, and thapsigargin. Moreover, adenosine enhanced accumulations of Ins(1,4,5)P3 that were significantly reduced by U-73122 and CPT. These data suggest that adenosine induces the release of ATP by activating an Ins(1,4,5)P3 sensitive-Ca2+ pathway through the stimulation of A1 receptors.

Adenosine↗

PubMed2005

Salt-sensitive hypertension, Na+/Ca2+ exchanger, and vascular smooth muscle.

Hypertension is the most common chronic disease and is the leading risk factor for death caused by stroke, myocardial infarction, and end-stage renal failure. The critical importance of excess salt intake in the pathogenesis of hypertension is widely recognized. However, the molecular mechanisms underlying salt-sensitive hypertension remain obscure. Recent studies using selective Na(+)/Ca(2+) exchanger (NCX) inhibitors and genetically engineered mice provide compelling evidence that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX type 1 (NCX1) in arterial smooth muscle. Cardiotonic steroids, such as endogenous ouabain, which may contribute to the pathogenesis of salt-sensitive hypertension, seem to be necessary for NCX1-mediated hypertension. These findings have enabled us to explain how high salt intake leads to hypertension and further to describe the potential of vascular NCX1 as a new therapeutic or diagnostic target for salt-sensitive hypertension.

Aniline Compounds↗

PubMed2005

Contribution of anaphylatoxins to allergic inflammation in human lungs.

The contribution of complement activation to allergic asthma remains controversial. In order to elucidate the role played by the complement split products, anaphylatoxins C3a and C5a, we evaluated their effects on production of cysteinyl-leukotrienes (cysLTs) by human lung fragments following an anaphylactic reaction. The lung tissues obtained from two patients with lung cancer showed C5aR-, C5L2R-, and C3aR-mRNA expression. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). The co-addition of human C5a at doses of 0.1 to 10 ng/ml to the anti-IgE antibody potentiated cysLT production. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1,000 ng/ml seemed to increase cysLT production, but not to any significant extent. A novel C5a receptor complementary peptide, acetylated peptide A, dose-dependently inhibited cysLT production by the human lung fragments following the anaphylactic reaction in the presence of 1 ng/ml C5a. However, this peptide did not inhibit cysLT production in the presence of 100 ng/ml C3a. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lung tissues and contributes to allergic inflammation in disorders such as asthma, thus acetylated peptide A may be useful for suppressing allergic inflammation in the lungs.

Anaphylatoxins↗

PubMed2005

Comparison of effects of nitric oxide synthase (NOS) inhibitors on plasma nitrite/nitrate levels and tissue NOS activity in septic organs.

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar-King rats. The rats treated with the three iNOS inhibitors (N-(3-(aminomethyl)benzyl)acetamidine (1400W), (1 S, 5 S, 6 R, 7 R )-2-aza-7-chloro-3-imino-5-methylbicyclo [4.1.0] heptane hydrochloride (ONO-1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose-dependent decreases in plasma NOx levels and NOS activity in the lungs. The non-selective NOS inhibitor (N(G)-methyl-L-arginine (L-NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.

Animals↗

PubMed2004

Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smooth muscle.

Excessive salt intake is a major risk factor for hypertension. Here we identify the role of Na(+)/Ca(2+) exchanger type 1 (NCX1) in salt-sensitive hypertension using SEA0400, a specific inhibitor of Ca(2+) entry through NCX1, and genetically engineered mice. SEA0400 lowers arterial blood pressure in salt-dependent hypertensive rat models, but not in other types of hypertensive rats or in normotensive rats. Infusion of SEA0400 into the femoral artery in salt-dependent hypertensive rats increases arterial blood flow, indicating peripheral vasodilation. SEA0400 reverses ouabain-induced cytosolic Ca(2+) elevation and vasoconstriction in arteries. Furthermore, heterozygous NCX1-deficient mice have low salt sensitivity, whereas transgenic mice that specifically express NCX1.3 in smooth muscle are hypersensitive to salt. SEA0400 lowers the blood pressure in salt-dependent hypertensive mice expressing NCX1.3, but not in SEA0400-insensitive NCX1.3 mutants. These findings indicate that salt-sensitive hypertension is triggered by Ca(2+) entry through NCX1 in arterial smooth muscle and suggest that NCX1 inhibitors might be useful therapeutically.

Alternative Splicing↗

PubMed2004

Comparison study between the mechanisms of allergic asthma amelioration by a cysteinyl-leukotriene type 1 receptor antagonist montelukast and methylprednisolone.

We investigated the effects of cysteinyl-leukotriene (cysLT) type 1 receptor antagonist montelukast (MK) and compared them with those of methylprednisolone (MP) in an allergic asthma model. Rats sensitized to ovalbumin (OVA) received repeated intratracheal exposure to OVA for up to 3 consecutive days. Pretreatment with MK or MP before OVA exposure inhibited late airway response (LAR) and reduced cellular infiltration into the bronchial submucosa after the triple OVA. The amount of N-acetyl-leukotriene E(4) in the bile was significantly reduced by pretreatment with MK or MP, suggesting that both drugs reduced the production of cysLTs in the lungs. In the in vitro study, when the fragments of lungs that had been repeatedly pretreated with MK or MP and exposed to OVA were removed and incubated with OVA, the coaddition of either drug significantly reduced cysLT production. In contrast, the cysLT production following the addition of OVA to the lung fragments that had not received in vivo pretreatment with either drug was inhibited by MK but not by MP. These results indicate that MK and MP inhibit LAR by suppressing the infiltration of inflammatory cells into the bronchial submucosa, thereby inhibiting the production of cysLTs in the lungs, and that MK but not MP may inhibit cysLT production directly. The different effects on cysLT production between the two drugs may provide a rationale for the use of combination therapy with cysLT(1) receptor antagonists and steroids for the treatment of asthma.

Acetates↗

PubMed2004

Endothelin-1 enhances the activity of Na+/Ca2+ exchanger type 1 in renal epithelial cells.

The Na+/Ca2+ exchanger, an ion transporter across the plasma membrane, is considered to play a role in calcium reabsorption in the renal nephron. We found that endothelin-1 enhanced Na+/Ca2+ exchange activity in renal epithelial LLC-PK1 cells. Treatment with endothelin-1 increased concomitantly the phosphorylation of Na+/Ca2+ exchanger type 1 (NCX1). Chelerythrine and prolonged exposure to phorbol 12-myristate 13-acetate abolished the activation of NCX1 induced by endothelin-1. To further analyze the activation mechanism of NCX1 by endothelin-1, we examined the effects of endothelin-1 in LLC-PK1 cells expressing NCX1 with mutated exchanger inhibitory peptide regions, which have either no Na+-dependent inactivation (XIP-4YW) or accelerated inactivation (F223E). The exchange activities in LLC-PK1 cells expressing wild-type NCX1 or F223E were stimulated by endothelin-1, but not in cells expressing XIP- 4YW. These results suggest that endothelin-1 activates NCX1 in renal epithelial cells through the pathway of protein kinase C and the process related to Na-dependent inactivation.

Animals↗

PubMed2004

Endothelin-1 aggravates hypoxia/reoxygenation-induced injury in renal epithelial cells through the activation of a Na+/Ca2+ exchanger.

We analyzed the role of the Na/Ca2+ exchanger (NCX) in hypoxia/reoxygenation-induced injury and also its interaction with endothelin-1 in the proximal epithelial cell line LLC-PK1. The hypoxia/reoxygenation protocol caused a significant leakage of lactate dehydrogenase from parental LLC-PK1 cells, which was markedly suppressed by KB-R7943, a selective NCX inhibitor. Overexpression of wild-type NCX1 into LLC-PK1 cells enhanced the release of lactate dehydrogenase and produced more severe morphological changes, such as bleb formation, during reoxygenation. Endothelin-1 significantly aggravated hypoxia/reoxygenation- induced injuries in parental and NCX1-overexpressing LLC-PK1 cells. Such aggravation by endothelin-1 was not observed in cells overexpressing a deregulated NCX1 mutant, which displays no protein kinase C-dependent activation. KB-R7943 suppressed those cell injuries aggravated by endothelin-1, but not those in cells overexpressing a KB-R7943-insensitive NCX1 mutant. We confirmed that these cell injuries were relevant to their cellular Ca accumulations induced by hypoxia/reoxygenation. These results suggest that Ca2+ overload via NCX plays a critical role in hypoxia/reoxygenation-induced renal tubular injury, and that endothelin-1 aggravates the cell damage through the activation of NCX.

Animals↗

PubMed2004

The exchanger inhibitory peptide region-dependent inhibition of Na+/Ca2+ exchange by SN-6 [2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a novel benzyloxyphenyl derivative.

We investigated the properties and interaction domains of SN-6 [2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester], a newly synthesized and selective Na(+)/Ca(2+) exchange (NCX) inhibitor. SN-6 (0.3-30 microM) inhibited preferentially intracellular Na(+)-dependent (45)Ca(2+) uptake (i.e., the reverse mode) compared with extracellular Na(+)-dependent (45)Ca(2+) efflux (i.e., the forward mode) in NCX1-transfected fibroblasts. SN-6 was 3- to 5-fold more inhibitory to (45)Ca(2+) uptake in NCX1 (IC(50) = 2.9 microM) than to that in NCX2 or NCX3 but not to that in NCKX2. We searched for regions that may form the SN-6 receptor by NCX1/NCX3-chimeric analyses and determined that amino acid regions 73 to 108 and 193 to 230 in NCX1 are mostly responsible for the differential drug response between NCX1 and NCX3. Further site-directed mutagenesis revealed that double substitutions of Val227 and Tyr228 in NCX1, which exist within the exchanger inhibitory peptide (XIP) region, mimicked the different drug response. In addition, F213R, G833C, and N839A mutations in NCX1 resulted in loss of drug sensitivity. Exchangers with mutated XIP regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had markedly reduced sensitivity or hypersensitivity to SN-6, respectively. Cell ATP depletion enhanced the inhibitory potency of SN-6. Therefore, SN-6 at lower doses (IC(50) = 0.63 microM) potently protected against hypoxia/reoxygenation-induced cell damage in renal tubular cells overexpressing NCX1, suggesting that this drug predominantly works under hypoxic/ischemic conditions. These properties of SN-6, which may be derived from its interaction with the XIP region, are advantageous to developing it as a new anti-ischemic drug.

Adenosine Triphosphate↗

PubMed2004

[The mechanism of ATP release as an autocrine/paracrine molecule].

Many studies have been performed to clarify the underlying mechanisms of the release of ATP as an autocrine / paracrine signaling molecule. So far, there is a variety of findings on the mode of release of this nucleotide. This review focused on the possible mechanisms of ATP release. The ATP binding cassette, especially CFTR (cystic fibrosis transmembrane conductance regulator), is a strong candidate for a channel or a transporter for outward movement of ATP. CFTR, which is activated via phosphorylation by protein kinase A, causes an opening of channels for Cl(-) and ATP(4-), releasing ATP. However, the possible involvement of CFTR in ATP release is still under dispute. As another candidate of the membrane machinery, the hemichannel of gap junction has been raised. Mechanical stress and photoliberation of caged InsP(3) induce the release of ATP as a paracrine through the hemichannel accompanied with the increase of [Ca(2+)]i. These events result in the Ca(2+)wave as cell-to-cell communications. In conclusion, an authoritative view of the mechanism of ATP release remains to be made clear in future studies.

Adenosine Triphosphate↗

PubMed2003

Molecular determinants of Na+/Ca2+ exchange (NCX1) inhibition by SEA0400.

SEA0400 is a potent and selective Na(+)/Ca(2+) exchanger (NCX) inhibitor. We evaluated the inhibitory effects of SEA0400 on Na(+)(i)-dependent (45)Ca(2+) uptake and whole-cell Na(+)/Ca(2+) exchange currents in NCX-transfected fibroblasts. SEA0400 preferentially inhibited (45)Ca(2+) uptake by NCX1 compared with inhibitions by NCX2, NCX3, and NCKX2. SEA0400 also selectively blocked outward exchange currents from NCX1 transfectants. We searched for regions that may form the SEA0400 receptor in the NCX1 molecule by NCX1/NCX3 chimeric analysis. The results suggest that the first intracellular loop and the fifth transmembrane segment are mostly responsible for the differential drug responses between NCX1 and NCX3. Further site-directed mutagenesis revealed that multiple mutations at Phe-213 markedly reduced sensitivity to SEA0400 without affecting that to KB-R7943. We also found that Gly-833-to-Cys mutation (within the alpha-2 repeat) greatly reduced the inhibition by SEA0400, but unexpectedly the NCX1 chimera with an alpha-2 repeat from NCKX2 possessed normal drug sensitivity. In addition, exchangers with mutated exchanger inhibitory peptide regions, which display either undetectable or accelerated Na(+)-dependent inactivation, had a markedly reduced sensitivity or hypersensitivity to SEA0400, respectively. To verify the efficacy of the NCX inhibitor, we examined the renoprotective effect of SEA0400 in a hypoxic injury model using porcine renal tubular cells. SEA0400 protected against hypoxia/reoxygenation-induced cell damage in tubular cells expressing wild-type NCX1 but not in cells expressing SEA0400-insensitive mutants. These results suggest that Phe-213, Gly-833, and residues that eliminate Na(+)-dependent inactivation are critical determinants for the inhibition by SEA0400, and their mutants are very useful for checking the pharmacological importance of NCX inhibition by SEA0400.

Alternative Splicing↗

PubMed2003

Contribution of capsaicin-sensitive sensory neurons to stress-induced increases in gastric tissue levels of prostaglandins in rats.

We examined whether capsaicin-sensitive sensory neurons might be involved in the increase in the gastric tissue level of prostaglandins, thereby contributing to the reduction of water immersion restraint stress (WIR)-induced gastric mucosal injury in rats. Gastric tissue levels of calcitonin gene-related peptide (CGRP), 6-keto-PGF1alpha, and PGE2 were transiently increased 30 min after WIR. These increases were significantly inhibited by subcutaneous injection of capsazepine (CPZ), a vanilloid receptor antagonist, and by functional denervation of capsaicin-sensitive sensory neurons induced by the administration of high-dose capsaicin. The administration of capsaicin (orally) and CGRP (intravenously) significantly enhanced the WIR-induced increases in the gastric tissue level of prostaglandins 30 min after WIR, whereas CGRP-(8-37), a CGRP receptor antagonist, significantly inhibited them. Pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of nitric oxide (NO) synthase (NOS), and that with indomethacin inhibited the WIR-induced increases in gastric tissue levels of prostaglandins, whereas either pretreatment with aminoguanidine (AG), a selective inhibitor of the inducible form of NOS, or that with NS-398, a selective inhibitor of cyclooxygenase (COX)-2, did not affect them. CPZ, the functional denervation of capsaicin-sensitive sensory neurons, and CGRP-(8-37) significantly increased gastric MPO activity and exacerbated the WIR-induced gastric mucosal injury in rats subjected to 4-h WIR. The administration of capsaicin and CGRP significantly increased the gastric tissue levels of prostaglandins and inhibited both the WIR-induced increases in gastric MPO activity and gastric mucosal injury 8 h after WIR. These effects induced by capsaicin and CGRP were inhibited by pretreatment with L-NAME and indomethacin but not by pretreatment with AG and NS-398. These observations strongly suggest that capsaicin-sensitive sensory neurons might release CGRP, thereby increasing the gastric tissue levels of PGI2 and PGE2 by activating COX-1 through activation of the constitutive form of NOS in rats subjected to WIR. Such activation of capsaicin-sensitive sensory neurons might contribute to the reduction of WIR-induced gastric mucosal injury mainly by inhibiting neutrophil activation.

Animals↗

PubMed2003

Induction of leukotriene C4 synthase after the differentiation of rat basophilic leukemia cells with retinoic acid and a low dose of actinomycin D and its suppression with methylprednisolone.

Leukotriene C(4) synthase (LTC(4) S) is a pivotal enzyme for generation of cysteinyl-leukotrienes (cysLTs). LTC(4) S activity in rat basophilic leukemia-1 (RBL-1) cells increased after culture in the presence of retinoic acid (RA) analogues, which was inhibited by cycloheximide or actinomycin D (ACD). Unexpectedly, the co-addition of a low dose of ACD with RA further potentiated the upregulation of the LTC(4) S activity. Daunorubicin and mitomycin C also had a similar effect. When stimulated with calcium ionophore A23187, control cells did not produce cysLTs, but RA-treated cells generated cysLTs and the co-addition of ACD further increased. While LTC(4) S mRNA and protein increased in the cells treated with RA, the co-addition of ACD further potentiated both in proportion to the LTC(4) S activity. The effect of ACD was considered to enhance the transcription rate of LTC(4) S gene, but not the mRNA-stability. The addition of methylprednisolone (MP) inhibited generation of cysLTs from the cells with A23187-stimulation and also did LTC(4) S activity, but did not inhibit 5-lipoxygenase (5-LOX). The suppression of LTC(4) S with MP showed a dependent manner on the time-point and duration of MP-treatment after RA-addition which was correlated with reduction in LTC(4) S mRNA and protein. The cells cultured with RA plus ACD contained more histamine, chymase activity, and granules in the cytoplasm than the control cells, suggesting differentiation to mature mast cells. In consideration of RA-differentiation therapy, it may be of pathophysiological relevance that the antineoplastic agents potentiate RA-induced, steroid-sensitive, induction of LTC(4) S in RBL-1 cells.

Animals↗

PubMed2002

Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release.

Intracellular signal transduction pathways involved in ATP release evoked by angiotensin II (Ang II) were investigated in cultured guinea pig Taenia coli smooth muscle cells. Ang II (0.3-1 microM) elicited substantial release of ATP from the cells, but not from a human fibroblast cell line. However, Ang II even at 10 microM failed to cause a leakage of lactate dehydrogenase (LDH) from the smooth muscle cells. The release of ATP by Ang II was suppressed by 10 microM SC52458, an AT1 receptor antagonist, not by 10 microM PD123319, an AT2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 microM U73122, a phospholipase C inhibitor, and 0.5 microM thapsigargin, a Ca2+-ATPase inhibitor. Furthermore, the release was hampered by 50 microM BAPTA/AM, an intracellular Ca2+ chelator, but not by 0.1 microM nifedipine, a voltage gated Ca2+ channel inhibitor. The basal release of ATP was increased by BAPTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca2+ signals activated via stimulation of Ins(1,4,5)P3 receptor are involved in the release of ATP evoked by Ang II.

Adenosine Triphosphate↗

PubMed2002

Measurement of dorsal root ganglion P2X mRNA by SYBR Green fluorescence.

The P2X receptor is a receptor-gated cationic channel that responds to ATP. The quantification of P2X mRNA expression in dorsal root ganglion (DRG) provides important information for neuropathic pain studies. We developed a rapid and sensitive external-standard-based real-time quantitative PCR assay for the quantification of mRNA of P2X receptors in mouse tissue samples. The assay uses a double-stranded DNA fluorescent dye, SYBR Green I, to continuously monitor product formation with a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). To establish the quantitative PCR amplification in a wide range of target transcripts, optimum parameters of primer sequences, concentrations of primers and/or templates, and PCR thermal protocols were experimentally determined. We also tested the reliability of this method in established experimental murine models, which were made by ligation or cutting down of the sciatic nerve. The parameters defined in this assay should be applicable to the quantification of other types of pain models and other tissue samples of mouse.

Animals↗