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In affinity are close to the ion-binding site and may, for instance, be involved in helixhelix interactions that are important for the integrity of the ion translocation pocket. One strength of the chimera approach is illustrated by the reciprocal relationship between kinetic parameters in the two chimeras. The kinetic changes observed when the first three transmembrane domains in sNKCC1 are converted to the human counterpart hs3.1 ; are mirrored when the same domain in hNKCC1 is changed to shark sh3.1 ; . Thus, the apparent affinities for Na and Rb are about half way between shark and human values in each of the chimeras, whereas the Cl affinities are only about 15% different from the parent species. There is some deviation from reciprocity with regard to Km Rb ; , but, nonetheless, the directionality of the change is as expected. These observations reassure us that we have used systematic and informative mutations that are not acting simply by disrupting the normal structure of the protein. In this paper we report Km values for ion transport and bumetanide inhibition. These parameters are related to actual binding site affinities in a complex manner that also depends on the rate constants in the transport cycle. Thus, it is theoretically possible that observed differences in Km values could be caused by changes in a translocation rate constant rather than to a change in a binding site affinity. Although the specific relationships are model dependent, in the general case one would expect to see parallel differences in apparent affinities for all three ions if this were a significant factor. In fact, we see substantial changes in Km cations ; but not in Km Cl ; , and we see individual effects of specific residue pairs on Km Na ; these results are reconcilable with simple kinetic models only if individual ion affinities are being changed. We conclude that the differences between mutants in this study result from the exchange of affinity-modifying residues between human and shark. The results of these studies provide a dramatic distinction between regions important in bumetanide binding and regions determining Cl affinity. Whereas Km Cl ; is changed 15% by modification of the residues in the first two transmembrane domains, about 80% of the species difference in Ki bumetanide ; is conferred by changes in that region. This is strong evidence against a hypothesis that the bumetanide-binding site is the same as the Cl-binding site, a popular idea 24 ; that had its origins in the competitive interaction of Cl in increasing Ki bumetanide ; 11, 20 ; . This does not rule out the possibility that part of the bumetanide-binding site is shared with the Cl site.
FIG. 1. Muscimol-induced intracellular Cl loss in immature neurons. Inset: Neurons were preincubated in normal HEPES MEM at 37C for 10 min and then equilibrated in HEPESMEM with 36Cl 0.4 Ci ml ; for 0 60 min. A: after a 30 min-equilibration with 36Cl 0.4 Ci ml ; , neurons were treated with HEPESMEM containing 36Cl 0.4 Ci ml ; either in the presence or absence of 30 M muscimol for 10 min. In the positive control experiment, cells were exposed to 10 mM [Cl ]o HEPESMEM plus 36Cl 0.4 Ci ml ; for 10 min. Data are means SE; n 5 6. * P 0.05 vs. control ; #P 0.05 vs. 36 muscimol-treated Bonferroni Dunn ; . B: neurons were treated with HEPESMEM containing Cl 0.4 Ci ml ; in the presence of 10 M bumetanide, 30 M muscimol, 30 M muscimol 10 M bicuculline, or 30 M muscimol 10 M bumetanide for 10 min. In the positive control experiments, cells were exposed to 75 mM containing 36Cl 0.4 Ci ml ; either in the presence or absence of 10 M bumetanide for 10 min. n 4 14. * P 0.05 vs. control; #P 0.05 vs. 75 mM [K Bonferroni Dunn.
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3 the abbreviations used are: egfr, epidermal growth factor receptor; pi3k, phosphatidylinositide 3 -kinase; pkb, protein kinase b; scid, severely combined immunodeficient; erk, extracellular signal-regulated kinase; met hgfr, hepatocyte growth factor receptor; ocip#, ontario cancer institute pancreas number; tunel, terminal deoxynucleotidyl transferase tdt ; -mediated nick end labeling.
PH 8 ; . Subsequent procedures were all at O-4 "C. The ghosts were pelleted by centrifugation at 10, 000 x g for 1 min SS-34 rotor ; and washed with an additional 20 volumes of LB, then resuspended in 0.3 volumes of LB and sonicatedfor 10 s with a probe sonifer Branson ; . Nuclei and residual nucleated ghosts were removed by centrifugation 1500 x g for 10 min ; . Crude membranes were then pelleted from the supernatant by high speed centrifugation 100, 000 X g for 50 min in a Beckman Ti-50.2 rotor ; . The pellets were resuspended in buffer A in mM, 250 sucrose, 1 EGTA, 0.1 phenylmethylsulfonyl fluoride, and 10 HEPES brought to pH 7.4 with triethanolamine ; and stored on ice or snap-frozen in liquid N2 and stored at -70 "C. Determination of PHlBumetanide Binding to Membranes-13H1 Bumetanide binding ; o membranes was measured by a filtration a&a; similar to that described by Forbush and Palfrey 1983 ; . The binding reactions were performed in a final volume of 0.1 ml. The membranes were diluted with buffer A to a protein concentration of 2.86 mg ml, as determined by the Folin reagent, and 0.07 ml 0.2 mg ; added to each binding reaction containing 0.03 ml of a mixture of the appropriate amount of salts, HEPES buffer, and 13H]bumetanide with or without unlabeled diuretic to give the desired final concentration of each. Unless stated otherwise the binding reaction contained the followine: in mM final concentration ; 175 sucrose. 10 HEPES. DH 7.4, 20 ga', 40 K', 20 Cl-, 20 SO: -, 6.7 EGTA, 0.67 phenylmetiylsulfonyl fluoride, and 120 nM [3H]bumetanide 0.79 &i ; + 10 unlabeled bumetanide. At various times the samples were diluted with 4 ml of ice-cold wash buffer 10 mM HEPES, pH 8.0 ; and filtered through Whatman GF-B filters on a 24-port filtration apparatus Brandel ; followed rapidly by three additional ice-cold 4-ml washes. Under these conditions dissociation of [3H]bumetanide from membranes was negligible cf. Fig. 36 ; . The filters were then placed in vials with 4 ml of Budgetsolve scintillation mixture RPI ; , and counted with an efficiency of -53%. The results are expressed in pmoles of [3H]bumetanide bound milligram of membrane protein. Determination of r3H]Bumetanide Binding to Who& Cells-The method of Haas and Forbush 1986 ; was used to determine [3H] bumetanide binding to whole cells.
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R. Simon: Dosis und Zeitabhngigkeit der hmodynamischen Toleranzentwicklung whrend intravenser Nitrattherapie beim akuten Myokardinfarkt. Z. Kardiol. 80 1991 ; , 279285. Metelitsa, V. I., S. Y. Martsevich, M. P. Kozyreva, I. D. Slastnikova: Enhancement of the efficacy of isosorbide dinitrate by captopril in stable angina pectoris. Amer. J rdiol. 69 1992 ; , 291296. Mikulic, E., J. A. Franciosa, J. N. Cohn: Comparative hemodynamic effects of chewable isosorbide dinitrate and nitroglycerin in patients with congestive heart failure. Circulation 52 1975 ; , 477482. Miller Jr, E. D, J. A. Ackerly, E. D. Vaughan Jr, M. J. Peach, R. M. Epstein: The renin-angiotensin system during controlled hypotension with sodium nitroprusside. Anesthesiology 47 1977 ; , 257262. Miller, L. F., B. H. Rumack: Clinical safety of high oral doses of N-acetylcysteine. Semin. Oncol. 10 1983 ; , 7681. Mitrovic, V., C. Gessner, P. Hain, K. D. Mller, M. Schlepper: Hemodynamic anti-ischemic and neurohumoral effects of slowrelease isosorbide-5-mononitrate in patients with coronary artery disease after short- and long-term therapy. Clin. Cardiol. 14 1991 ; , 209218. Mohiuddin, I. H., H. Kambara, T. Ohkusa, R. Nohara, T. Fudo, S. Ono, N. Tamaki, H. Ohtani, Y. Yonekura, C. Kawai, J. Konishi: Clinical evaluation of cardiac function by ambulatory ventricular scintigraphic monitoring VEST ; : Validation and study of the effects of nitroglycerin and nifedipine in patients with and without coronary artery disease. Amer. Heart J. 123 1992 ; , 386394. Muiesan, G., E. Agabiti-Rosei, L. Muiesan, G. Romanelli, P. Pollavini, C. Pasotti, G. Fiori, L. Muratori, A. M. Zuarini, C. Pastorini, S. Borziani, L. B. Bozzi, S. Marchetti: A multicenter trial of transdermal nitroglycerin in exercise-induced angina: Individual antianginal response after repeated administration. Amer. Heart J. 112 1986 ; , 233238. Muiesan, M. L., E. Agabiti-Rosei, G. Romanelli, M. Beschi, M. Castellano, M. Cefis, B. Cerrri, G. Pollavini, G. Muiesan: Transdermal nitroglycerin efficacy in patients with chronic stable angina pectoris as related to sympathetic and reninangiotensinaldosteron activity. Europ. Heart J. 13 1992 ; , 1521. Mnzel, T., T. Heitzer, S. Kurz, D. G. Harrison, C. Luhmann, L. Pape, M. Olschewski, H. Just: Dissociation of coronary vascular tolerance and neurohormonal adjustments during long-term nitroglycerin therapy in patients with stable coronary artery disease. J. Amer. Coll. Cardiol. 27 1996 ; , 297303. Mnzel, T., J. Holtz, A. Mlsch, D. J. Stewart, E. Bassenge: Nitrate tolerance in epicardial arteries or in the venous system is not reversed by N-acetylcysteine in vivo but tolerance-independent interactions exist. Circulation 79 1989 ; , 188197. Mnzel, T., H. Sayegh, B. A. Freeman, M. M. Tarpey, D. G. Harrison: Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J. clin. Invest. 95 1995 ; , 187194. Mnzel, T., D. J. Steward, J. Holtz, E. Bassenge: Preferential venoconstriction by cyclooxygenase inhibition in vivo without attenuation of nitroglycerin venodilation. Circulation 78 1988 ; , 407415. Murrell, W.: Nitro-glycerine as a remedy for angina pectoris. Lancet 1 1879 ; , 8081. Nabel, E. G., J. Barry, M. B. Rocco, K. Mead, A. P. Selwyn: Effects of dosing intervals on the development of tolerance to high dose transdermal nitroglycerin. Amer. J. Cardiol. 63 1989 ; , 663-669. Needleman, P., B. Jakschik, E. M. Johnson Jr: Sulfhydryl requirement for relaxation of vascular smooth muscle. J. Pharmacol. exp. Ther. 187 1973 ; , 324331. Needleman, P., E. M. Johnson: Mechanism of tolerance development to organic nitrates. J. Pharmacol. exp. Ther. 184 1973 ; , 709715. 203. Niestegge, H., G.Cordes, G. Blmchen: Toleranzentwicklung unter kontinuierlicher Nitroglycerin-Infusion. Herz 14 1989 ; , 6670. 204. Nyberg, G.: Klinische Erfahrungen mit einem Isosorbidmononitrat in Duriles-Galenik Coleb- Duriles ; . In: Grobecker, H., et al. Hrsg. ; : Chronische Nitrattherapie. Toleranz Retardierung Stellenwert. Verlag fr angewandte Wissenschaften, Mnchen 1987, S. 8194. 205. Odenthal, H. J., H. W. Wiechmann, W. Josephs, P. Lenga: Transdermale Nitroglycerinsysteme mit kontinuierlicher und diskontinuierlicher Substanzfreisetzung. Dtsch. med. Wschr. 116 1991 ; , 241247. 206. Ohlmeier, H., H. M. Mertens, M. Mller, H. Mannebach, U. Gleichmann: Vereinfachte Langzeittherapie der koronaren Herzkrankheit mit 120 mg retardiertem Isosorbiddinitrat einmal tglich. Untersuchung zur Wirkdauer und Toleranzentwicklung. Z. Kardiol. 75, Suppl. 3 1986 ; , 5056. 207. Olivari, M., P. Carlyle, B. S. Levine, J. Cohn: Hemodynamic and hormonal response to transdermal nitroglycerin in normal subjects and in patients with congestive heart failure. J. Amer. Coll. Cardiol. 2 1983 ; , 872888. 208. Olsson, G., J. Allgn, O. Amtorp, G. Nyberg, J. O. Parker: Absence of pre-dose rebound phenomena with once daily 5ISMN in a controlled-release formulation. Europ. Heart J. 13 1992 ; , 814817. 209. Osterspey, A., W. Jansen, M. Tauchert, V. Schell, M. Fuchs, V. Hombach, H. H. Hilger: Hmodynamische Wirkung von Molsidomin bei Akutgabe und Langzeitmedikation. Med. Welt Stuttg. ; 34 1983 ; , 523528. 210. Osterspey, A., W. Jansen, T. Ulbrich, P. Simon, M. Tauchert, H. H. Hilger: Wirkung von Nitroglycerinpflastern auf Hmodynamik und Belastbarkeit von Patienten mit koronarer Herzkrankheit. Dtsch. med. Wschr. 109 1984 ; , 714717. 211. Paciaroni, E., C. Luca: Discontinous transdermal nitroglyerin as treatment for stable angina in the elderly: a double-blind multicentre study. Europ. Heart J. 12 1991 ; , 10761080. 212. Packer, M., S. S. Gottlieb, P. D. Kessler, W. H. Lee, M. L. Kukin, N. Medina, M. Yushak: Overnight withdrawal of nitroglycerin therapy does not prevent the development of nitrate tolerance in severe heart failure. J. Amer. Coll. Cardiol. 11 1988 ; , 43A. 213. Packer, M., W. H. Lee, P. D. Kessler, S. S. Gottlieb, N. Medina, M. Yushak: Prevention and reversal of nitrate tolerance in patients with congestive heart failure. New Engl. J. Med. 317 1987 ; , 799804. 214. Packer, M., N. Medina, M. Yushak, W. H. Lee: Hemodynamic factors limiting the response to transdermal nitroglycerin in severe chronic congestive heart failure. Amer. J. Cardiol. 57 1986 ; , 260267. 215. Packer, M., J. Meller, N. Medina, M. Yushak, R. Gorlin: Determinants of drug responses in severe chronic heart failure. Activation of vasoconstrictor forces during vasodilatory therapy. Circulation 64 1981 ; , 506514. 216. Palmer, R. M. J., A. G. Ferrige, S. Moncada: Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature Lond. ; 327 1987 ; , 524526. 217. Panzenbeck, M. J., A. Baez, G. Kaley: Nitroglycerin and nitroprusside increase coronary blood flow in dogs by a mechanism independent of prostaglandin release. Amer. J. Cardiol. 53 1984 ; , 936940. 218. Parker, J. D., B. Farrell, T. Fenton, J. O. Parker: Effects of diuretic therapy on the development of tolerance during continuous therapy with nitroglycerin. J. Amer. Coll. Cardiol. 20 1992 ; , 616622. 219. Parker, J. D., A. B. Parker, B. Farrell, J. O. Parker: Effects of diuretic therapy on the development of tolerance to nitroglycerin and exercise capacity in patients with chronic stable angina. Circulation 93 1996 ; , 691696. 220. Parker, J. D., J. O. Parker: Effect of therapy with an angiotensin.
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The pulmonary carcinoma cells were grown in 24-well cluster plates for 0 or 60 min in EMEM containing 5.3 mmol L K ; , without control ; or with amphotericin B 3 mg L ; alone or combined with bumetanide 100 mol L ; , and then incubated for 15 min in the same medium supplemented with 1 mol L 86RbCl 500 Ci mol ; , with or without ouabain 100 mol L ; , alone or in combination. The concentrations of the channel-blocking agents were chosen because of the total inhibition of Na , K , 2Cl cotransport and Na , K ATPase activity.9 The cells were rinsed, trypsinized and transferred to scintillation vials, and radioactivity was determined by liquid scintillation counting. Isotope influx in drug-treated cells was expressed as a percentage of the influx of controls basal influx ; handled in parallel and without test substances. Counting ensured that the number of cells treated with drugs at 60 min were not reduced in number compared with untreated control cells. From a previous study9 we learned that three types of potassium flux pathway exist in this pulmonary carcinoma cell line: Na , K , 2Cl cotransport blocked by bumetanide ; , Na , K ATPase activity blocked by ouabain ; and high conductance K channels blocked by tetraethyl and butorphanol.
Concomitant reduction in cell Cl content. To test this, we measured HMTC Cl content after exposure of the cells to 100 M NA for 30 min Fig. 8 ; . We also measured cell Cl content after exposure of HTMC to either 10 M bumetanide alone or 10 M bumetanide plus 100 M NA for 30 min, as a means of determining whether the predicted reduction of cell Cl by 100 M NA was additive, with the expected reduction of cell Cl after Na-K-Cl cotransport inhibition. Figure 8A shows that exposure of HTMC to either 10 M bumetanide or 100 M NA for 30 min caused a significant reduction in cell Cl content, 14% and 11%, respectively. When HTMC were exposed to both 10 M bumetanide and 100 M NA for 30 min, cell Cl content was reduced in an additive manner, by 34%. These findings support the hypothesis that NA opens a Cl channel in HTMC, causing Cl efflux and reduction of cell Cl content. We also investigated the effects of bumetanide and NA on HTMC volume and found that changes in cell volume reflect changes in cell Cl content occurring after exposure of the cells to these agents Fig. 8B ; . Here bumetanide 10 M ; and NA 100 M ; separately reduced cell volume by 12% and 11%, respectively, whereas together they produced an additive cell shrinkage of 21%. These data support the hypothesis that NA, at concentrations of 100 M and lower, open a Cl channel in HTMC, promoting Cl efflux and reducing cell Cl content and cell volume.
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Banasiski, A.: Ubezpieczenia gospodarcze. op.cit. s. 53. Akty prawne pochodzily z 30 stycznia 1947 roku - pierwszy byl to dekret o uregulowaniu ubezpiecze rzeczowych i osobowych Dz. U. Nr 5, poz. 23 ; , drugi, rwnie dekret, dotyczyl Powszechnego Zakladu Ubezpiecze Wzajemnych Dz. U. Nr 19, poz. 79 ; Za: Ubezpieczenia gospodarcze. Red. T. Sangowski, op.cit. s. 363. 4 Ustawa z dnia 28 marca 1952 r. o ubezpieczeniach pastwowych Dz. U. Nr 20, poz. 130 ; . 5 Ustawa z dnia 2 grudnia 1958 r. o ubezpieczeniach majtkowych i osobowych Dz. U. Nr 72, poz. 357 ; . 6 Przepisy ustawy w tym zakresie uregulowano dopiero w 1972 roku rozporzdzeniem Rady Ministrw i dotyczyly wylcznie porednikw PZU Rozporzdzenie Rady Ministrw z 10 marca 1972 r. w sprawie porednictwa ubezpieczeniowego. Dz. U. Nr 9, poz. 59 ; . 7 Ustawa z dnia 1 grudnia 1961 r. Kodeks morski Dz. U. Nr 58 ; Tekst jedn. Dz. U. z 1986 r. Nr 22 , poz. 122 z pon. zm. 8 Ustawa z 23 kwietnia 1964 r. Kodeks cywilny Dz. U. Nr 16, poz. 93 ; . 9 Ustawa z dnia 20 wrzenia 1984r. o ubezpieczeniach majtkowych i osobowych Dz. U. Nr 30, poz. 160 ; . 10 Ustawa z dnia 17 maja 1989 r. o nowelizacji ustawy o ubezpieczeniach majtkowych i osobowych Dz. U. Nr 45, poz. 242 ; . 11 Ustawa z dnia 28 lipca 1990 r. o dzialalnoci ubezpieczeniowej Dz. U. Nr 59, poz. 944.
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Rapid and reversible, and to reach a defined equilibrium position Nakatsugawa et al., 1980 ; . Most other investigations relating to hepatocyte binding have focused on kinetic studies of uptake and metabolism of compounds by hepatocytes in which active uptake processes were expected to be present; for example, with drugs known to have extensive biliary clearance. The kinetics of uptake of benzylpenicillin and cefpiramide by rat hepatocytes has been found to occur through both a saturable transport mechanism and a passive process, whereas that of cefazolin takes place purely through a passive process Tsuji et al., 1986 ; . Other compounds with both active and passive hepatocyte uptake mechanisms include glycyrrhizin Ishida et al., 1993 ; , iodipamide Joppen et al., 1985 ; , tyramine Zhong et al., 1993 ; , pravastatin Yamazaki et al., 1993 ; , and bumetanide Petzinger et al., 1989 ; . The purpose of the current study is to determine the extent of hepatocyte binding for a set of drugs that exhibit a range of physicochemical properties, and to determine how the binding to hepatocytes is related to the physicochemical properties of the molecules and campral
Target Organ Damage: LV hypertrophy ECG, echocardiogram, or chest X-ray ; Proteinuria or elevated plasma creatinine men 1.34-1.6 mg dl i.e. 118-140 mol l, women 1.25-1.45 mg dl i.e. 110-128 mol l ; Ultrasound or radiological evidence of atherosclerotic plaque aortic, carotid, iliac, or femoral ; Generalized or focal narrowing of retinal arteries Associated Clinical Conditions: Cerebrovascular disease: ischemic stroke, cerebral hemorrhage, or TIA ; . Heart disease: MI, angina, coronary revascularization, or CHF ; . Renal disease: diabetic nephropathy or renal failure i.e. creatinine men 1.6 mg dl i.e. 140 mol l, women 1.45 mg dl i.e. 128 mol l ; . Vascular disease: dissecting aortic aneurysm or symptomatic arterial disease ; Advanced hypertensive retinopathy: hemorrhages, exudates, or papilledema ; . Other Factors Adversely Influencing Prognosis but not used for Risk Stratification: Micro-albuminuria in diabetic patient Impaired glucose tolerance Sedentary lifestyle Raised fibrinogen High risk socio-economic group High risk ethnic group High risk geographic region and bumetanide.
Article, publication date, and citation information can be found at : dmd etjournals . doi: 10.1124 dmd.105.004838. The online version of this article available at : dmd etjournals ; S contains supplemental material and camptosar.
Loop diuretics: the site of action of frusemide, bumetanide and torasemide is at the thick ascending loop of henle, where they inhibit the action of the nkcc2 cotransporter fig 1 ; , resulting in sodium, potassium and chloride wasting with diuresis.
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