(1/428) Identification of selective mechanism-based inactivators of cytochromes P-450 2B4 and 2B5, and determination of the molecular basis for differential susceptibility.

Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.  (+info)

(2/428) Guest exchange in an encapsulation complex: a supramolecular substitution reaction.

Encapsulation complexes are reversibly formed assemblies in which small molecule guests are completely surrounded by large molecule hosts. The assemblies are held together by weak intermolecular forces and are dynamic: they form and dissipate on time scales ranging from milliseconds to days-long enough for many interactions, even reactions, to take place within them. Little information is available on the exchange process, how guests get in and out of these complexes. Here we report that these events can be slow enough for conventional kinetic studies, and reactive intermediates can be detected. Guest exchange has much in common with familiar chemical substitution reactions, but differs in some respects: no covalent bonds are made or broken, the substrate is an assembly rather than a single molecule, and at least four molecules are involved in multiple rate-determining steps.  (+info)

(3/428) Eukaliuric diuresis and natriuresis in response to the KATP channel blocker U37883A: micropuncture studies on the tubular site of action.

1. Systemic application of U37883A, a blocker of ATP sensitive potassium (KATP) channels, elicits diuresis and natriuresis without significantly altering urinary potassium excretion. 2. To elucidate tubular sites of action upstream to the distal nephron, micropuncture experiments were performed in nephrons with superficial glomeruli of anaesthetized Munich-Wistar-Fromter rats during systemic application of U37883A (1, 5 or 15 mg kg-1 i.v.). 3. The observed eukaliuric diuresis and natriuresis in response to U37883A at 15 mg kg-1 was accompanied by an increase in early distal tubular flow rate (VED) from 10 - 18 nl min(-1) reflecting a reduction in fractional reabsorption of fluid up to this site (FR-fluid) of 13%. The latter proposed an effect on water-permeable segments such as the proximal tubule which could fully account for the observed reduction in fractional reabsorption of Na+ up to the early distal tubule (FR-Na+) of 8% and the increase in early distal tubular Na+ concentration ([Na+]ED) from 35 - 51 mM whereas [K+]ED was left unaltered. 4. In comparison, furosemide (3 mg kg-1 i.v.), which acts in the water-impermeable thick ascending limb, elicited diuresis, natriuresis and kaliuresis which were associated with a fall in FR-Na+ of 10% with no change in FR-fluid, and a rise in [Na+]ED from 42 - 117 mM and [K+]ED from 1.2 - 5.7 mM with no change in VED. 5. Direct late proximal tubular fluid collections confirmed a significant inhibition of fluid reabsorption in proximal convoluted tubule in response to systemic application of U37883A. 6. These findings suggest that the diuretic and natriuretic effect upstream to the distal tubule in response to systemic application of U37883A involves actions on water-permeable segments such as the proximal convoluted tubule.  (+info)

(4/428) Analysis of vasoconstrictor responses to histamine in the hindlimb vascular bed of the rabbit.

Hemodynamic responses to histamine were investigated in the anesthetized rabbit. Intravenous injections of histamine induced dose-dependent decreases in systemic arterial pressure that were blocked by the H(1)-receptor antagonist pyrilamine but not the H(2) antagonist cimetidine. Injections of histamine and the H(1) agonist 6-[2-(4-imidazolyl)ethylamine]-N-(4-trifuormethylphenyl)-heptan ecardo xamide dimaleate (HTMT) into the hindlimb perfusion circuit increased hindlimb perfusion pressure, whereas the H(2) agonist dimaprit decreased perfusion pressure and the H(3)-receptor agonist R-(-)-alpha-methylhistamine did not alter perfusion pressure. Pyrilamine reduced hindlimb vasoconstrictor responses to histamine and HTMT but did not alter vasodilator responses to dimaprit. Cimetidine reduced the response to dimaprit but did not alter vasoconstrictor responses to histamine or HTMT. The H(3)-receptor antagonist thioperamide was without effect on responses to the histamine agonists. These data suggest the presence of H(1) and H(2) receptors and that histamine for the most part acts by stimulating H(1) receptors to produce vasoconstriction in the hindlimb vascular bed of the rabbit. Responses to histamine, HTMT, and norepinephrine were significantly enhanced by a nitric oxide synthase inhibitor at a time when vasodilator responses to dimaprit were unaltered and responses to acetylcholine were significantly reduced. Responses to histamine and the H(1) and H(2) agonists were not affected by the cyclooxygenase inhibitor meclofenamate or by ATP-sensitive K(+) channel, alpha-adrenergic, or angiotensin AT(1) receptor antagonists. The present data suggest that H(1) receptors mediate both systemic vasodepressor and hindlimb vasoconstrictor responses to histamine.  (+info)

(5/428) Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A.

1. A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced. 2. Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co-injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone. 3. K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 microM glibenclamide. K+-current carried by hKir6.1+SUR1 was inhibited by the putative vascular-selective KATP channel inhibitor U37883A (IC50 32 microM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected. 4. The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore-forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels.  (+info)

(6/428) Inhibition of vascular K(ATP) channels by U-37883A: a comparison with cardiac and skeletal muscle.

1 The aim of this study was to investigate the selectivity of the ATP-sensitive potassium (K(ATP)) channel inhibitor U-37883A (4-morpholinecarboximidine-N-1-adamantyl-N'-1-cyclohexyl). Membrane currents through K(ATP) channels were recorded in single muscle cells enzymatically isolated from rat mesenteric artery, cardiac ventricle and skeletal muscle (flexor digitorum brevis). K(ATP) currents were induced either by cell dialysis with 0.1 mM ATP and 0.1 mM ADP, or by application of synthetic potassium channel openers (levcromakalim or pinacidil). 2 U-37883A inhibited K(ATP) currents in smooth muscle cells from rat mesenteric artery. Half inhibition of 10 microM levcromakalim-induced currents occurred at a concentration of 3.5 microM. 3 Relaxations of rat mesenteric vessels caused by levcromakalim were reversed by U-37883A. 1 microM levcromakalim-induced relaxations were inhibited at a similar concentration of U-37883A (half inhibition, 1.1 microM) to levcromakalim-induced KATP currents. 4 K(ATP) currents activated by 100 microM pinacidil were also studied in single myocytes from rat mesenteric artery, skeletal muscle and cardiac ventricle. 10 microM U-37883A substantially inhibited K(ATP) currents in vascular cells, but had little effect in skeletal or cardiac myocytes. Higher concentrations of U-37883A (100 microM) caused a modest decrease in K(ATP) currents in skeletal and cardiac muscle. The sulphonylurea K(ATP) channel antagonist glibenclamide (10 microM) abolished currents in all muscle types. 5 The effect of U-37883A on vascular inward rectifier (KIR) and voltage-dependent potassium (KV) currents was also examined. While 10 microM U-37883A had little effect on these currents, some inhibition was apparent at higher concentrations (100 microM) of the compound. 6 We conclude that U-37883A inhibits K(ATP) channels in arterial smooth muscle more effectively than in cardiac and skeletal muscle. Furthermore, this compound is selective for K(ATP) channels over KV and KIR channels in smooth muscle cells.  (+info)

(7/428) Early increases in renal kallikrein secretion on administration of potassium or ATP-sensitive potassium channel blockers in rats.

1 This study aimed to examine whether administration of potassium or ATP-sensitive potassium channel (KATP channel) blockers caused early increases in renal kallikrein (KK) secretion. To clarify this mechanism, the effect on renal KK secretion of a KATP channel blocker was compared with the effect resulting from use of an osmotic diuretic or volume load. Furthermore, the effect on potassium-induced increases in renal KK secretion by an additional treatment using a KATP channel blocker was examined. Lastly, the effect of a KATP channel blocker on renal KK secretion was also examined in superfused slices of kidney cortex. 2 Intravenous infusion of potassium augmented renal KK secretion within 30 min while urine volume increased gradually in both the potassium loading and control groups. 3 Administration of the KATP channel blocker, 4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexylhydr ochloride (PNU-37883A) or glibenclamide, caused a dose-dependent increase in renal KK secretion. 4 The concentration of KK in urine was higher in the PNU-37883A group as compared to the osmotic-diuretic or volume-load group. 5 PNU-37883A had no additive effect on the potassium-induced increase in renal KK secretion. 6 Renal KK secretion increased in slices of kidney cortex incubated with PNU-37883A within 10 min of superfusion. 7 In conclusion, administration of both potassium and KATP channel blockers induced early increases in renal KK secretion in the absence of the washout phenomenon. Potassium loading may have increased renal KK secretion through the same mechanism as the KATP channel blocker.  (+info)

(8/428) Effects of the bcr/abl kinase inhibitors AG957 and NSC 680410 on chronic myelogenous leukemia cells in vitro.

The tyrphostin AG957 (NSC 654705) inhibits p210bcr/abl, the transforming kinase responsible for most cases of chronic myelogenous leukemia (CML). The present studies were performed to determine the fate of AG957-treated cells and assess the selectivity of AG957 for CML myeloid progenitors. When K562 cells (derived from a patient with blast crisis CML) were treated with AG957, dose- and time-dependent p210bc/abl down-regulation was followed by mitochondrial release of cytochrome c, activation of caspase-9 and caspase-3, and apoptotic morphological changes. These apoptotic changes were inhibited by transfection with cDNA encoding dominant negative caspase-9 but not dominant-negative FADD or blocking anti-Fas antibodies. In additional experiments, a 24-h AG957 exposure caused dose-dependent inhibition of K562 colony formation in soft agar. To extend these studies to clinical samples of CML, peripheral blood mononuclear cells from 10 chronic phase CML patients and normal controls were assayed for the growth of hematopoietic colonies in vitro in the presence of increasing concentrations of AG957. These assays demonstrated selectivity of AG957 for CML progenitors, with median IC50s (CML versus normal) of 7.3 versus >20 microM AG957 in granulocyte colony-forming cells (P < 0.001), 5.3 versus >20 microM in granulocyte/macrophage colony-forming cells (P < 0.05), and 15.5 versus > 20 microM in erythroid colony-forming cells (P > 0.05). The adamantyl ester of AG957 (NSC 680410) down-regulated p210bcr/abl in K562 cells and inhibited granulocyte colony formation in CML specimens at lower concentrations without enhanced toxicity in normal progenitors. These observations not only demonstrate that AG957-induced p210bcr/abl down-regulation is followed by activation of the cytochrome c/Apaf-1/caspase-9 pathway but also indicate that this class of kinase inhibitor exhibits selectivity worthy of further evaluation.  (+info)

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