Our library of drug research abstracts drawn from the medical literature is updated on a regular schedule, and you can be assured that new bumex research articles will be listed here shortly after becoming available to us.
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Carbonic anhydrase inhibitors. Sulfonamide diuretics revisited-old leads for new applications?
Org Biomol Chem. 2008 Jul 21; 6(14): 2499-2506
Temperini C, Cecchi A, Scozzafava A, Supuran CT
Sulfonamide diuretics such as hydrochlorothiazide, hydroflumethiazide, quinethazone, metolazone, chlorthalidone, indapamide, furosemide and bumetanide were tested as inhibitors of the zinc enzyme carbonic anhydrases (CAs, EC 4.2.1.1). These drugs were discovered in a period when only isoform CA II was known and considered physiologically/pharmacologically relevant. We prove here that although acting as moderate to weak inhibitors of CA II, all these drugs considerably inhibit other isozymes known nowadays to be involved in critical physiologic processes, among the 16 CAs present in vertebrates. Some low nanomolar/subnanomolar inhibitors against such isoforms were detected, such as among others metolazone against CA VII, XII and XIII, chlorthalidone against CA VB, VII, IX, XII and XIII, indapamide against CA VII, IX, XII and XIII, furosemide against CA I, II and XIV, and bumethanide against CA IX and XII. The X-ray crystal structure of the CA II-indapamide adduct was also resolved at high resolution, and the binding of this sulfonamide to the enzyme was compared to that of dichlorophenamide, sulpiride and a pyridinium containing sulfonamide. Indapamide binds to CA II in a manner not seen earlier for any other CA inhibitor, which might be important for the design of compounds with a different inhibition profile.
J Neurosci. 2008 Jul 2; 28(27): 6996-7005
Shulga A, Thomas-Crusells J, Sigl T, Blaesse A, Mestres P, Meyer M, Yan Q, Kaila K, Saarma M, Rivera C, Giehl KM
A shift of GABA(A)-mediated responses from hyperpolarizing to depolarizing after neuronal injury leads to GABA(A)-mediated increase in [Ca2+](i). In addition, central neurons become dependent on BDNF for survival. Whether these two mechanisms are causally interrelated is an open question. Here, we show in lesioned CA3 hippocampal neurons in vitro and in axotomized corticospinal neurons in vivo that posttraumatic downregulation of the neuron-specific K-Cl cotransporter KCC2 leads to intracellular chloride accumulation by the Na-K-2Cl cotransporter NKCC1, resulting in GABA-induced [Ca2+](i) transients. This mechanism is required by a population of neurons to survive in a BDNF-dependent manner after injury, because blocking GABA(A)-depolarization with the NKCC1 inhibitor bumetanide prevents the loss of neurons on BDNF withdrawal. The resurgence of KCC2 expression during recovery coincides with loss of BDNF dependency for survival. This is likely mediated through BDNF itself, because injured neurons reverse their response to this neurotrophin by switching the BDNF-induced downregulation of KCC2 to upregulation.
Neuroscience. 2008 Jun 10;
Fournier S, Kinkead R
The present study used an in vitro brainstem preparation from pre-metamorphic tadpoles and adult bullfrogs (Lithobates catesbeiana) to understand the neural mechanisms associated with central O(2) chemosensitivity and its maturation. In this species, brainstem hypoxia increases fictive lung ventilation in tadpoles but decreases in adults. Previous studies have shown that alpha(1)-adrenoceptor inactivation prevents these responses, suggesting that noradrenergic neurons are involved. We first tested the hypothesis that the pons (which includes noradrenergic neurons from the locus coeruleus; LC) plays a role in the lung burst frequency response to central hypoxia by comparing the effects of brainstem transection at the LC level between pre-metamorphic tadpoles and adults. Data show that brainstem transection prevents the lung burst frequency response in both stage groups. During development, the progressive decrease in the Na(+)/K(+)/Cl(-) co-transporter NKCC1 contributes to the maturation of neural networks. Because NKCC1 becomes activated during hypoxia, we then tested the hypothesis that NKCC1 contributes to maturation of the central O(2) chemoreflex. Double labeling experiments showed that the proportion of tyrosine hydroxylase positive neurons expressing NKCC1 in the LC decreases during development. Inactivation of NKCC1 with bumetanide bath application reversed the lung burst response to hypoxia in tadpoles. Bumetanide inhibited the response in adults. These data indicate that a structure within the pons (potentially the LC) is necessary to the central hypoxic chemoreflex and demonstrate that NKCC1 plays a role in central O(2) chemosensitivity and its maturation in this species.
BMC Neurosci. 2008 Jun 30; 9(1): 57
Diykov D, Turchinovich A, Zoidl G, Hoffmann KP
ABSTRACT: BACKGROUND: During development the switch from a depolarizing to a hyperpolarizing action of GABA is a consequence of a decrease of the Na+-K+-2Cl- co-transporter (NKCC1, Cl--uptake) and increase of the K+-Cl- co-transporter (KCC2, Cl--extrusion) expression. However albino visual cortex neurons don't show a corresponding decrease in intracellular chloride concentration during development of the visual system as compared to pigmented animals. RESULTS: Our study revealed that more cells express NKCC1 in albinos compared to pigmented rat visual cortex neurons whereas KCC2 is expressed in all cells in both strains. We determined a positive relationship between the presence of NKCC1 and an inhibitory deficit in single neurons of the albino visual cortex. After pharmacological blockade of NKCC1 function with its specific inhibitor, bumetanide, the reversal potential of electrically evoked GABAAR-mediated postsynaptic currents and, as a consequence, [Cl-]i in albino visual cortex neurons shifted to the pigmented rat brain value. In conclusion, our pharmacological experiments and subsequent single cell real time PCR analysis of the co-transporter mRNA demonstrated that the inhibitory deficit present in the albino visual cortical network is almost exclusively mediated by NKCC1. CONCLUSIONS: Our findings suggest that blocking of NKCC1 in albino visual cortex neurons could improve processing in visual cortex and therefore might be beneficial for vision in albinos.
Talanta. 2008 Jun 30; 76(1): 15-20
Zheng X, Lu M, Zhang L, Chi Y, Zheng L, Chen G
A simple and sensitive online field-amplification sample stacking (FASS) pre-enrichment method following by capillary electrophoresis with amperometric detection has been developed for the determination of diuretics, such as indapamide (IDP), hydrochlorothiazide (HCT) and bumetanide (BMTN) in urine. Under the optimum conditions, it was found that the low concentration buffer solution could be used as the diluents for simultaneous field-amplification injection of three diuretics after electrokinetically injecting a short water plug (15 kV, 3 s). Three analytes could be well separated within 10 min in an uncoated fused-silica capillary with H(3)BO(3)-Na(2)B(4)O(7) (BB) buffer solution (pH 8.98). The detection limits (S/N=3) were 9.0 ng/mL for IDP, 20 ng/mL for HCT and 1.5 ng/mL for BMTN, respectively. The detection limits of three diuretics were much lower by FASS than that by conventional sample injection, of which the detection limits were 340, 890 and 330 ng/mL for IDP, HCT and BMTN, respectively. Especially, for bumetanide the detection limit was 220-time lower by FASS. The linear ranges of three diuretics were all over three orders of magnitude. The proposed method has been successfully applied to analyze the diuretics in human urine samples without off-column sample pre-concentration.
Regulation of endothelin-1 expression and function by nutrient stress in mouse colon epithelia.
Scand J Gastroenterol. 2008; 43(7): 886-94
Kozakai T, Sakate M, Saida K
OBJECTIVE: The endothelin (ET) system is influenced by a variety of stress conditions in many tissues. However, the effects of nutrient stress conditions on ET expression and its function are not well understood in the intestinal tract, while ET-1 gene expression and peptide were found in the intestinal tract. The aim of this study was to investigate the effect of feeding and fasting on the expression of ET-1 and short-circuit current (Isc) induced by ET-1 in mouse colon. MATERIAL AND METHODS: Mice were fed freely, fasted for 48 h, and re-fed after fasting, respectively. ET-1 mRNA levels and peptide concentrations were analyzed using real-time polymerase chain reaction (PCR) and sandwich ELISA, respectively. Isc of epithelial tissue was measured under short-circuit conditions using a Ussing chamber. RESULTS: ET-1 mRNA expression and peptide concentrations in epithelial colonic tissue were significantly increased 48 h after fasting, and decreased within 2 h of re-feeding after a 48-h fast. Furthermore, the addition of ET-1 to the serosal but not the mucosal side increased Isc in colonic epithelia. An increase in Isc was caused by chloride ion (Cl(-)) secretion because Isc induced by ET-1 was blocked by bumetanide and Cl(- -) free conditions. In addition, an increase in Isc induced by ET-1 in colon excised from fasted mice was much lower than that obtained from free-fed mice. CONCLUSIONS: Gene expression, peptide concentration, and the function of ET-1 in mouse colonic epithelia are regulated by nutrient stress.
RENAL NA-K-CL COTRANSPORTER ACTIVITY AND VASOPRESSIN-INDUCED TRAFFICKING ARE LIPID RAFT-DEPENDENT.
Am J Physiol Renal Physiol. 2008 Jun 25;
Welker P, Boehlick A, Mutig K, Salanova M, Kahl T, Schlueter H, Blottner D, Ponce-Coria J, Gamba G, Bachmann S
Apical bumetanide-sensitive Na(+)-K(-)-2Cl(-)-cotransporter (NKCC2), the kidney-specific member of a cation-chloride cotransporter superfamily, is an integral membrane protein responsible for the transepithelial reabsorption of NaCl. The role of NKCC2 is essential for renal volume regulation. Vasopressin (AVP) controls NKCC2 surface expression in cells of the thick ascending limb of the loop of Henle (TAL). We found that 40-70% of Triton X-100-insoluble NKCC2 was present in cholesterol-enriched lipid rafts (LR) in rat kidney and cultured TAL cells. The related Na(+)Cl(-)-cotransporter, NCC, from rat kidney was distributed in LR as well. NKCC2-containing LR were detected both intracellularly and in the plasma membrane. Bumetanide-sensitive transport of NKCC2 as analyzed by 86Rb+ influx in Xenopus laevis oocytes was markedly reduced by methyl-beta-cyclodextrin (MbetaCD)-induced cholesterol depletion. In TAL, short term AVP application induced apical vesicular trafficking along with a shift of NKCC2 from non-raft to LR fractions. In parallel, increased colocalization of NKCC2 with the LR ganglioside GM1 and their polar translocation were assessed by confocal analysis. Apical biotinylation showed twofold increases in NKCC2 surface expression. These effects were blunted by mevalonate-lovastatin/MbetaCD-induced cholesterol deprivation. Collectively, these findings demonstrate that a pool of NKCC2 distributes in rafts. Results are consistent with a model in which LR mediate polar insertion, activity, and AVP-induced trafficking of NKCC2 in the control of transepithelial NaCl transport. Key words: thick ascending limb, lipid raft, Xenopus oocyte, cholesterol depletion.
J Biol Chem. 2008 Jun 11;
Smith L, Smallwood N, Altman A, Liedtke CM
Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a PKCdelta-mediated intracellular signalling pathway. Downregulation of PKCdeltaprevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signalling pathway identified coimmunoprecipitation of PKCdelta with with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCdeltaactivates SPAK which subsequently activates NKCC1 through phosphorylation. Double stranded silencing RNA directed against SPAK reduced SPAK protein expression by 65.8% and prevented increased phosphorylation of NKCC1 and functional activation of NKCC1 during hyperosmotic stress, measured as bumetanide-sensitive basolateral to apical (86)Rb flux. Using recombinant proteins, we demonstrate direct binding of PKCdelta to SPAK, PKCdelta-mediated activation of SPAK, binding of SPAK to the amino terminus of NKCC1 (NT-NKCC1, aa 1-286), and competitive inhibition of SPAK-NKCC1 binding by a peptide encoding a SPAK binding site on NT-NKCC1. The carboxyl terminus of SAPK (aa 316-548) pulls down endogenous NKCC1 from Calu-3 total cell lysates and GST-tagged NT-NKCC1 pulls down endogenous SPAK. In intact cells, hyperosmotic stress increased phosphorylated PKCdelta, indicating activation of PKCdelta, and activity of endogenous SPAK kinase. Inhibition of PKCdelta activity with rottlerin blocked the increase in SPAK kinase activity. The results indicate that PKCdelta acts upstream of SPAK to increase activity of NKCC1 during hyperosmotic stress..
Capillary electrophoresis of diuretics and probenecid in methanol.
J Chromatogr A. 2008 Jul 11; 1198-1199: 215-9
Sirén H, Shimmo R, Sipola P, Abenet S, Riekkola ML
Actual mobilities and dissociation constants of six diuretics (benzthiazide, bumetanide, ethacrynic acid, furosemide, hydrochlorothiazide and chlorothiazide) and probenecid were investigated in methanol by capillary zone electrophoresis (CZE). The actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH(*) of the methanolic background electrolyte (BGE) solution. The measurement of effective mobilities was carried out mainly by a pressure-mediated capillary electrophoresis (CE) method. For comparison, parallel measurements were carried for two of the analytes by the conventional capillary electrophoresis approach, without pressure. The pK(a)(*) values in methanol were calculated by non-linear curve fitting to the measured mobility values. The difference between the pK(a) values in water and methanol was about 5 units for the loop diuretics (furosemide, bumetanide, ethacrynic acid) and probenecid and around 3-4 units for the thiazide diuretics. Knowledge of the ionisation behaviour of compounds in methanol paves the way for the wider use of methanol as background electrolyte solvent in capillary electrophoresis. Moreover, as is demonstrated in the current study, the calculation of actual mobilities and pK(a) values facilitates the optimisation of pH conditions for the separation, thereby applications can be expanded, and the combination of capillary electrophoresis with electrospray ionisation mass spectrometry becomes easier.
AAPS J. 2008 Jun 4;
Morris ME, Felmlee MA
The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1-4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1-4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including gamma-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and L: -lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.