Expression of hepatocyte growth factor and its receptor in the placental basal plate in pre-eclamptic pregnancies.
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Adult↗
Biomedical subjects
H Y Ahn
Publications and source records attributed to H Y Ahn.
Involvement of phospholipase D in oxidative stress-induced necrosis of vascular smooth muscle cells.
Phospholipase D (PLD) has been associated with necrosis. However, it is not clear whether PLD plays a causative role in this cellular process. We investigated the role of PLD in oxidative stress-induced necrosis of vascular smooth muscle cells (VSMCs). Pervanadate (hydrogen peroxide plus orthovanadate) but not hydrogen peroxide alone activated PLD in a dose- and time-dependent manner. Exposure of VSMCs to pervanadate resulted in necrosis. Pretreatment with butan-1-ol, a PLD inhibitor, attenuated both pervanadate-induced necrosis and increase of intracellular Ca(2+). Removal of extracellular Ca(2+) inhibited pervanadate-induced necrosis by 50%. These results suggest that PLD activation mediates pervanadate-induced necrosis of VSMCs, which is at least partly due to Ca(2+) toxicity.
Animals↗
Endothelium-dependent vasorelaxant and antiproliferative effects of apigenin.
This study was designed to determine whether the relaxant effect of apigenin was endothelium dependent and to examine the possible antiproliferative effect of apigenin. Apigenin relaxed the phenylephrine-precontracted endothelium-intact aortic rings with IC(50) value of 3.7+/-0.5 microM and removal of a functional endothelium significantly attenuated this relaxation (IC(50)=8.2+/-0.9 microM). However, apigenin did not affect the 0.1 microM phorbol 12,13-dibutyrate-induced contraction (IC(50)=34.6+/-1.2 microM) within the concentration range that relaxed the phenylephrine-contracted arteries, suggesting that apigenin did not influence protein kinase C-mediated contractile mechanisms in rat aorta. Pretreatment of apigenin significantly potentiated the relaxant effect of acetylcholine on phenylephrine-induced contraction. Pretreatment with N(G)-nitro-L-arginine methyl ester (L-NAME) or methylene blue reduced the relaxant effect of apigenin. Apigenin (10 microM) increased the guanosine 3',5'-cyclic monophosphate (cGMP) content of endothelium-intact tissues. Pretreatment with L-NAME (100 microM) or removal of endothelium significantly suppressed the effect of apigenin on cGMP production. In addition, apigenin significantly inhibited [3H]thymidine incorporation into DNA of primary cultured rat aortic smooth muscle cell in a dose-dependent manner. These findings suggest that besides influx and release of Ca(2+), nitric oxide (NO) and cGMP may account for the apigenin-induced endothelium-dependent relaxation and hypotensive activity. Both vasorelaxant and antiproliferative activities may contribute to a benefit of apigenin in the vascular system.
Animals↗
Bypassing the kinetic trap of serpin protein folding by loop extension.
The native form of some proteins such as strained plasma serpins (serine protease inhibitors) and the spring-loaded viral membrane fusion proteins are in a metastable state. The metastable native form is thought to be a folding intermediate in which conversion into the most stable state is blocked by a very high kinetic barrier. In an effort to understand how the spontaneous conversion of the metastable native form into the most stable state is prevented, we designed mutations of alpha1-antitrypsin, a prototype serpin, which can bypass the folding barrier. Extending the reactive center loop of alpha1-antitrypsin converts the molecule into a more stable state. Remarkably, a 30-residue loop extension allows conversion into an extremely stable state, which is comparable to the relaxed cleaved form. Biochemical data strongly suggest that the strain release is due to the insertion of the reactive center loop into the major beta-sheet, A sheet, as in the known stable conformations of serpins. Our results clearly show that extending the reactive center loop is sufficient to bypass the folding barrier of alpha1-antitrypsin and suggest that the constrain held by polypeptide connection prevents the conversion of the native form into the lowest energy state.
Binding Sites↗
Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).
Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.
3T3 Cells↗
Draft guidance for industry extended-release solid oral dosage forms. Development, evaluation and application of in vitro-in vivo correlations.
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Administration, Oral↗
Cyclic AMP and cyclic GMP relax phorbol ester-induced contractions of rat aorta by different mechanisms.
This study was designed to test the hypothesis that 8-Br-cAMP and 8-Br-cGMP dependent relaxation of phorbol dibutyrate stimulated contractions of intact rat aorta are independent of changes in the level of myosin light chain phosphorylation. Phorbol dibutyrate stimulated contraction with a concomitant increase in myosin light chain phosphorylation in normal tissues and without an increase in myosin light chain phosphorylation in calcium-depleted tissues. Phorbol dibutyrate stimulated contractions in normal CaCl2-containing physiological salt solution were relaxed in a concentration-dependent manner by 8-Br-cAMP and 8-Br-cGMP. Phorbol dibutyrate-induced contractions in the absence of Ca2+ were only relaxed by 8-Br-cGMP; 8-Br-cAMP had no effect. The relaxation induced by 8-Br-cGMP was associated with a decrease in myosin light chain phosphorylation suggesting that cGMP-dependent protein kinase may alter the activity of either the myosin light chain kinase or phosphatase. The relaxation induced by 8-Br-cAMP was not associated with a decrease in phosphorylation suggesting that cAMP-dependent protein kinase may uncouple myosin light chain phosphorylation from force.
8-Bromo Cyclic Adenosine Monophosphate↗
Vasodilating and hypotensive effects of fangchinoline and tetrandrine on the rat aorta and the stroke-prone spontaneously hypertensive rat.
Comparative studies of the effects of tetrandrine (TET) and fangchinoline (FAN), two major components of the Radix of Stephannia tetrandrae, on vasodilations and on calcium movement in vascular smooth muscle, and studies of hypotensive effects on stroke-prone spontaneously hypertensive rats (SHRSP) were performed in the following experiments. TET and FAN inhibited high K+ (65.4 mM) and induced sustained contraction in the rat aorta smooth muscle strips. IC50 values for TET and FAN were 0.27 +/- 0.05 microM (n = 6) and 9.53 +/- 1.57 microM (n = 6), respectively, and this inhibition was antagonized by increasing the Ca2+ concentration in the medium. The IC50 of TET for norepinephrine (NE)-induced contraction (0.86 +/- 0.04 g) was 3.08 +/- 0.05 microM (n = 4), and the IC50 of FAN for NE-induced contraction (0.88 +/- 0.07 g) was 14.20 +/- 0.40 microM (n = 4). At the molecular level, radiolabelled 45Ca2+ uptake tests revealed that TET and FAN also inhibited high K+ (65.4 mM) and 1 microM NE-stimulated Ca2+ influx in rat aorta strips at the maximal concentration was needed to inhibit the contraction. TET (3 mg/kg) and FAN (30 mg/kg) administered by intravenous (i.v.) bolus injection also lowered the mean arterial pressure (MAP) significantly during the period of observation in conscious SHRSP, respectively. These results showed that TET was more potent than FAN in blocking calcium channels and antihypertensive activity.
Alkaloids↗
Resolution of the enantiomers of ibuprofen; comparison study of diastereomeric method and chiral stationary phase method.
In this study, an indirect diastereomeric method and a direct method utilizing a chiral stationary phase (CSP) were investigated for the resolution of ibuprofen enantiomers. In the indirect method, ethylchloroformate (ECF) and 2-ethoxy-1-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) were utilized as first-step derivatizing reagents in acetonitrile or toluene. In the direct CSP method, ibuprofen enantiomers were derivatized to p-nitrobenzyl ureides and then resolved on an (R)-(-)-(1-naphthyl)ethylurea CSP column. The derivatization procedure took place in 10 min with an overall inversion efficiency of 90.3%. Racemization was not observed under the derivatization conditions used. The HPLC-CSP method was utilized to study the pharmacokinetics of ibuprofen enantiomers in dog plasma after a single oral administration of 200 mg of ibuprofen racemate.
Animals↗
A hypothesis for the mechanism of receptor and G-protein-dependent enhancement of vascular smooth muscle myofilament Ca2+ sensitivity.
Agonist activation enhances smooth muscle myofilament Ca2+ sensitivity. The increased force accompanying receptor stimulation (over Ca2+ alone) requires GTP and is reversed by GDP beta S, demonstrating a G-protein dependence. Protein kinase C (PKC) activators, such as phorbol esters, mimic and PKC inhibitors block the agonist-induced increase in Ca2+ sensitivity, suggesting a role for PKC in the regulation of Ca2+ sensitivity. Myosin light chain (MLC) phosphorylation levels are transiently increased by agonist stimulation, but steady-state levels of MLC phosphorylation are similar to those in response to Ca2+ alone. Thus, G-protein-mediated inhibition of MLC phosphatase may account for the initial increase in force development but not the increase in steady-state force. In contrast to MLC, calponin phosphorylation levels are maintained during agonist stimulation of intact vascular smooth muscle. We propose that stimulation of smooth muscle by membrane depolarization increases MLC phosphorylation, but as a result of inhibition by unphosphorylated calponin only a portion of the phosphorylated cross bridges attach to actin. Agonist stimulation produces the same steady-state level of MLC phosphorylation but also leads to calponin phosphorylation via a PKC-dependent pathway. Thus, during agonist stimulation, all phosphorylated cross bridges can interact with actin, thereby generating significantly greater levels of force.
Actin Cytoskeleton↗
Transient myosin phosphorylation at constant Ca2+ during agonist activation of permeabilized arteries.
Norepinephrine (NE) plus guanosine triphosphate (GTP) increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized smooth muscle. We used alpha-toxin-permeabilized rabbit mesenteric arteries to determine the temporal relationships among force, myosin light chain (MLC) phosphorylation, stiffness, and shortening velocity during contractions in response to Ca2+ alone and to the same [Ca2+] in the presence of NE plus GTP. The addition of NE plus GTP caused a marked increase in the tonic contraction but only transiently elevated the level of MLC phosphorylation over that observed in the presence of Ca2+ alone. NE plus GTP induced similar increases in force and stiffness, but shortening velocity depended solely on the [Ca2+]. A regulated MLC phosphatase could explain the initial increase in force and MLC phosphorylation, but not the maintenance of enhanced force while MLC phosphorylation levels fell to values similar to those in response to Ca2+ alone. Therefore, additional elements must be involved in the maintenance of the receptor and G protein-dependent increase in myofilament Ca2+ sensitivity.
Animals↗
Endothelin increases myofilament Ca2+ sensitivity in alpha-toxin-permeabilized rabbit mesenteric artery.
This study was designed to investigate the mechanism of endothelin-1 (ET-1) contractions in Staphylococcus alpha-toxin-permeabilized vascular smooth muscle. Rabbit small mesenteric arteries permeabilized with alpha-toxin were mounted for isometric or isotonic force recording or were processed for determination of myosin light chain (MLC) phosphorylation levels. Addition of 100 nM ET-1 plus 10 microM GTP significantly enhanced myofilament Ca2+ sensitivity as compared with the addition of Ca2+ alone (EC50, 0.47 microM Ca2+ for Ca2+ alone and 0.13 microM Ca2+ for ET-1 plus (GTP). This enhanced sensitivity was reversed by GDP beta S. ET-1-induced contractions were relaxed at a constant [Ca2+] by the addition of 30 microM cAMP or cGMP, demonstrating a direct effect of the cyclic nucleotides on contractile regulation. Inhibition of protein kinase C activity by 100 nM staurosporine relaxed ET-1 plus GTP-induced contractions, and pretreatment with 40 microM chelerythrine inhibited the ET-1 plus GTP increase in force. At 0.32 microM Ca2+, steady-state levels of shortening velocity were not increased by ET-1 plus GTP, although steady-state levels of MLC phosphorylation were significantly enhanced. The ET-1-induced increase in MLC phosphorylation was not altered by changes in [Ca2+], whereas the shortening velocity was Ca2+ dependent, suggesting that the increase MLC phosphorylation level may be the result of protein kinase C, rather than MLC kinase, activation. These results are consistent with the hypothesis that ET-1 increases myofilament Ca2+ sensitivity by a G protein-dependent pathway and subsequent activation of protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)
Actin Cytoskeleton↗
Dibutyryl cyclic AMP and forskolin inhibit phosphatidylinositol hydrolysis, Ca2+ influx and contraction in vascular smooth muscle.
Dibutyryl cyclic AMP and forskolin inhibited the contraction induced by norepinephrine (NE) more strongly than the high K(+)-induced contraction in isolated rat aorta. These inhibitors inhibited the 45Ca2+ influx stimulated by NE but not that by high K+, and they inhibited NE-induced inositol monophosphate accumulation. These results suggest that cAMP inhibits NE-induced contraction, at least partly, by inhibiting the alpha-adrenoceptor-mediated signal transduction and high K(+)-induced contraction by decreasing Ca2+ sensitivity but not Ca2+ influx.
Animals↗
Stereoselective systemic disposition of ibuprofen enantiomers in the dog.
The pharmacokinetics of ibuprofen are complicated by the unidirectional metabolic inversion of the (-)-R- to (+)-S-enantiomer. Chiral inversion is of therapeutic significance since the drug's pharmacologic activity has been shown to depend upon the (+)-S-isomer. As a result, the present study was undertaken to determine if chiral inversion occurs systemically and to elucidate further the kinetics of the inversion process. Experiments were performed in the beagle dog after intravenous bolus injections of ibuprofen enantiomers separately [100 mg (-)-R, n = 4; 100 mg (+)-S, n = 4] and as admixtures of varying proportions [100 mg (-)-R + 100 mg (+)-S, n = 4; 100 mg (-)-R + 200 mg (+)-S, n = 2]. Plasma samples of (-)-R- and (+)-S-enantiomers were measured by a stereospecific HPLC assay after all drug administrations. Based on the area under the plasma concentration-time curves for (+)-S after administration of each enantiomer alone, chiral inversion was 70 to 75%. A progressive reduction in total plasma clearance of (-)-R-ibuprofen is also observed as increasing amounts of (+)-S-enantiomer are added to the system. The results demonstrate that chiral inversion occurs to a significant extent in the systemic circulation in dog and that R-to-S inversion of ibuprofen may be inhibited by its (+)-S-enantiomer.
Animals↗
Stereoselective disposition of ibuprofen enantiomers in the isolated perfused rat kidney.
The renal clearance of ibuprofen enantiomer was studied separately in the isolated perfused rat kidney at initial perfusate concentrations of 10 micrograms/ml (n = 4) and 100 micrograms/ml (n = 4). Perfusate and urine samples were measured for R(-) and S(+)-ibuprofen using a stereospecific HPLC assay; urine samples were also analyzed after alkaline hydrolysis. Functional viability of the kidney was assured by determining the fractional excretion of glucose and glomerular filtration rate (GFR) at similar perfusion pressures. The clearance of ibuprofen was equivalent to the apparent formation clearance of conjugated enantiomer since unchanged ibuprofen could not be detected in the urine. At 10 and 100 micrograms/ml, the clearance (+/- SD) of R(-)-ibuprofen was 2.50 +/- 1.28 and 2.19 +/- 1.42 microliters/min, respectively. At 100 micrograms/ml, the clearance of S(+)-ibuprofen was 0.805 +/- 0.290 microliters/min. The protein binding of ibuprofen was found to be concentration dependent and favored the R(-)-enantiomer. The excretion ratio (clearance corrected for free fraction and GFR) of R(-)-ibuprofen was 0.398 +/- 0.209 and 0.295 +/- 0.209 for perfusate concentrations of 10 and 100 micrograms/ml, respectively. The excretion ratio of S(+)-ibuprofen was 0.0886 +/- 0.0335 for perfusate concentrations of 100 micrograms/ml. These results demonstrate that the sum of renal mechanisms involved for the clearance of R(-)- and S(+)-ibuprofen was net reabsorption. Ibuprofen was recovered in the urine solely as conjugated material and no evidence of R(-) to S(+) conversion was observed. In addition, the data suggest that R(-)-ibuprofen is cleared through the kidney faster than its S(+)-enantiomer.
Animals↗
Synthesis and secretion of mucous glycoprotein by the gill of Mytilus edulis. I. Histochemical and chromatographic analysis of [14C]glucosamine bioincorporation.
The ability of the isolated gill epithelium of Mytilus edulis to incorporate [14C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins was investigated. Localization of mucous cells in the gill filament was achieved using histochemical staining techniques. Mucus cells containing neutral and acidic mucins were found in the lateral region, whereas mucus cells containing primarily neutral or sulfated mucins were found in the abfrontal region. Autoradiographic results showed that in both regions, the mucous cells were rich in content of the incorporated radiolabel. The secreted glycoproteins containing the incorporated radiolabel were analyzed by column chromatography using Bio-Gel P-2 and P-6. Two populations of the glycoproteins differing in molecular size were isolated. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of the high molecular weight protein, about 70% of the radiolabel and 85% of the carbohydrate content were removed from the protein. The alkaline borohydride cleavage resulted in the formation of at least six oligosaccharide chains of various lengths of sugar units. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contain N-acetylglucosamine, N-acetylgalactosamine, and galactose, fucose, and mannose as the neutral monosaccharides. The above results indicate that the isolated gill epithelium of M. edulis is capable of incorporating [14C]glucosamine in the synthesis of secretable mucin-type glycoproteins.
Animals↗
Inhibitory effects of caffeine on contractions and calcium movement in vascular and intestinal smooth muscle.
1. The mechanism of the inhibitory effect of caffeine was investigated using vascular smooth muscle of rabbit aorta and intestinal smooth muscle of taenia isolated from guinea-pig caecum. 2. Caffeine, 0.5-10 mM, relaxed the sustained contraction induced by 65.4 mM KCl or 10(-6) M noradrenaline in aorta, and by 45.4 mM KCl or 10(-6) M carbachol in taenia. The inhibitory effect of caffeine on the high K+-induced contraction was antagonized by external Ca2+ but not by the Ca2 channel activators, Bay K 8644 (10(-7) M) or CGP 28,392 (10(-7) M). Forskolin (2 x 10(-7) M) potentiated the inhibitory effect of caffeine on the noradrenaline-induced contraction but not on the high K+- or carbachol-induced contraction. Caffeine induced a time- and concentration-dependent increase in the cyclic AMP content of aorta and forskolin caused a further augmentation. 3. 45Ca2+ uptake was increased by high K+ or noradrenaline in aorta and by high K+ or carbachol in taenia. The increments were inhibited by caffeine at concentrations needed to inhibit muscle contractions. 4. 45Ca2+ in the cellular releasable site in aorta was decreased either by noradrenaline or by caffeine. Simultaneous application of noradrenaline and caffeine did not induce an additive decrease. 5. In aorta treated with a Ca2+-free solution, caffeine induced only a small contraction. Noradrenaline induced a greater contraction which was inhibited by caffeine. After washout of caffeine and noradrenaline, the second application of noradrenaline induced a transient contraction suggesting that caffeine does not deplete the noradrenaline-sensitive store. 6. It was concluded that caffeine has multiple sites of action in smooth muscle. Caffeine releases Ca2+ from a store which is apparently not sensitive to noradrenaline. Caffeine may inhibit noradrenalineinduced Ca2' release. Caffeine itself induces only a small contraction possibly because it decreases the Ca2+ sensitivity of contractile filaments and/or increases Ca2+ extrusion. Further, caffeine seems to inhibit stimulated Ca2+ influx. Cyclic AMP may be only partly responsible for the inhibitory effect of caffeine.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Inhibitory effects of procaine on contraction and calcium movement in vascular and intestinal smooth muscles.
1. The effects of procaine on muscle tension and 45Ca2+ movements were investigated in vascular smooth muscle of the rabbit aorta and intestinal smooth muscle of the taenia isolated from guinea-pig caecum. 2. Procaine (10 mM) induced a contraction in the taenia but had little effect on the resting tension in the aorta. 3. Procaine, 0.5-10 mM, relaxed the sustained contractions induced by 65.4 mM KCl and 10(-6) M noradrenaline in the aorta, and by 45.4 mM KCl, 10(-6) M carbachol and 10(-6) M histamine in the taenia. The inhibitory effect of procaine on the high K+-induced contractions was antagonized by external Ca2+ but not by the Ca2+ channel activators, Bay K 8644 and CGP 28,392. 4. 45Ca2+ uptake was increased by high K+ or noradrenaline in the aorta and by high K+ or carbachol in the taenia. The increments were inhibited by procaine at the concentrations needed to inhibit the muscle contractions. 5. In a Ca2+-free solution, noradrenaline and caffeine induced a transient contraction in the aorta, whereas a second application of each stimulant was almost ineffective. Addition of 1-10 mM procaine shortly before the first application of the stimulant inhibited the contraction. After washing the muscle with a Ca2+-free solution without procaine, the second application of the stimulant induced a greater contraction than that in control muscle without procaine pretreatment. 6. Noradrenaline and caffeine released 45Ca2+ from a cellular site in the aorta. Procaine inhibited the effects of these stimulants. 7. It was concluded that procaine may inhibit both the opening of Ca2+ channels and the release of Ca2 + from cellular stores and the former but not the latter effect may be attributable to a local anaesthetic action.
Animals↗