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Calcium-activated and voltage-gated potassium channels of the pancreatic islet impart distinct and complementary roles during secretagogue induced electrical responses.
Jacobson DA, Mendez F, Thompson M, Torres J, Cochet O, Philipson LH
(2010) J Physiol 588: 3525-37
MeSH Terms: Aminoquinolines, Animals, Calcium, Electrophysiological Phenomena, Evoked Potentials, Glucose, Humans, Hypoglycemic Agents, Imines, Insulin-Secreting Cells, Mice, Mice, Inbred C57BL, Mice, Knockout, Potassium Channels, Calcium-Activated, Potassium Channels, Voltage-Gated, Quinolines, Tolbutamide
Show Abstract · Added February 12, 2015
Glucose-induced β-cell action potential (AP) repolarization is regulated by potassium efflux through voltage gated (Kv) and calcium activated (K(Ca)) potassium channels. Thus, ablation of the primary Kv channel of the β-cell, Kv2.1, causes increased AP duration. However, Kv2.1(-/-) islet electrical activity still remains sensitive to the potassium channel inhibitor tetraethylammonium. Therefore, we utilized Kv2.1(-/-) islets to characterize Kv and K(Ca) channels and their respective roles in modulating the β-cell AP. The remaining Kv current present in Kv2.1(-/-) β-cells is inhibited with 5 μM CP 339818. Inhibition of the remaining Kv current in Kv2.1(-/-) mouse β-cells increased AP firing frequency by 39.6% but did not significantly enhance glucose stimulated insulin secretion (GSIS). The modest regulation of islet AP frequency by CP 339818 implicates other K(+) channels, possibly K(Ca) channels, in regulating AP repolarization. Blockade of the K(Ca) channel BK with slotoxin increased β-cell AP amplitude by 28.2%, whereas activation of BK channels with isopimaric acid decreased β-cell AP amplitude by 30.6%. Interestingly, the K(Ca) channel SK significantly contributes to Kv2.1(-/-) mouse islet AP repolarization. Inhibition of SK channels decreased AP firing frequency by 66% and increased AP duration by 67% only when Kv2.1 is ablated or inhibited and enhanced GSIS by 2.7-fold. Human islets also express SK3 channels and their β-cell AP frequency is significantly accelerated by 4.8-fold with apamin. These results uncover important repolarizing roles for both Kv and K(Ca) channels and identify distinct roles for SK channel activity in regulating calcium- versus sodium-dependent AP firing.
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17 MeSH Terms
The direct and indirect effects of insulin on hepatic glucose production in vivo.
Cherrington AD, Edgerton D, Sindelar DK
(1998) Diabetologia 41: 987-96
MeSH Terms: Animals, Blood Glucose, C-Peptide, Diabetes Mellitus, Type 2, Fatty Acids, Nonesterified, Glucose, Humans, Hypoglycemic Agents, Insulin, Liver, Reference Values, Tolbutamide
Added December 10, 2013
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12 MeSH Terms
Reconstitution of recombinant cytochrome P450 2C10(2C9) and comparison with cytochrome P450 3A4 and other forms: effects of cytochrome P450-P450 and cytochrome P450-b5 interactions.
Yamazaki H, Gillam EM, Dong MS, Johnson WW, Guengerich FP, Shimada T
(1997) Arch Biochem Biophys 342: 329-37
MeSH Terms: Animals, Aryl Hydrocarbon Hydroxylases, Chlorzoxazone, Cytochrome P-450 CYP2C9, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System, Cytochromes b5, Escherichia coli, Ethanolamines, Humans, Hydroxylation, Mixed Function Oxygenases, Mutation, NADP, Nifedipine, Oxidation-Reduction, Phospholipids, Recombinant Proteins, Testosterone, Theophylline, Tolbutamide, Warfarin
Show Abstract · Added May 26, 2014
Tolbutamide methyl hydroxylation and S-warfarin 7-hydroxylation activities were reconstituted in systems containing recombinant human cytochrome P450 (P450 or CYP) 2C10(2C9) and the optimal conditions for the systems were compared with those of bufuralol 1'-hydroxylation by CYP1A1, theophylline 8-hydroxylation by CYP1A2, bufuralol 1'-hydroxylation by CYP2D6, chlorzoxazone 6-hydroxylation by CYP2E1, and testosterone 6 beta-hydroxylation by CYP3A4. CYP2C10 required cytochrome b5 (b5) for optimal rates of tolbutamide and S-warfarin oxidations and b5 could be replaced by apo-b5; apo-b5 and b5 effects on the reconstituted systems have already been reported in systems containing CYP3A4 for the oxidation of testosterone and nifedipine and for the rapid reduction of CYP3A4 by NADPH-P450 reductase (H. Yamazaki et al., 1996, J. Biol. Chem. 271, 27438-27444). Stopped-flow studies, however, suggested that apo-b5 as well as b5 did not cause stimulation of the reduction of CYP2C10 by NADPH-P450 reductase, while the reduction rates were dependent on the substrates in reconstituted systems. Chlorzoxazone 6-hydroxylation by CYP2E1 was stimulated by b5, but not by apo-b5, in reconstituted systems. Neither apo- nor holo-b5 increased bufuralol 1'-hydroxylation activity by CYP1A1 or 2D6 or theophylline 8-hydroxylation by CYP1A2. Interestingly, we found that testosterone 6 beta-hydroxylation by CYP3A4 was stimulated by CYP1A2 (and also by a modified form in which the first 36 residues of the native human protein were removed) and CYP1A1 as well as by b5, and such stimulations were not seen when other P450 proteins (e.g., CYP2C10, 2D6, or 2E1) were added to the reconstituted systems. In contrast, substrate oxidations by CYP2C10 and CYP2E1 were not stimulated by other P450 proteins. The present results suggest that there are differences in optimal conditions for reconstitution of substrate oxidations by various forms of human P450 enzymes, and in some P450-catalyzed reactions protein-protein interactions between P450 and b5 and other P450 proteins are very important in some oxidations catalyzed by CYP2C10, 2E1, and 3A4.
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22 MeSH Terms
Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation activities in livers of Japanese and Caucasian populations.
Inoue K, Yamazaki H, Imiya K, Akasaka S, Guengerich FP, Shimada T
(1997) Pharmacogenetics 7: 103-13
MeSH Terms: Aryl Hydrocarbon Hydroxylases, Asian Continental Ancestry Group, Cytochrome P-450 CYP2C19, Cytochrome P-450 CYP2C9, Cytochrome P-450 Enzyme System, European Continental Ancestry Group, Genotype, Humans, Hydroxylation, Mephenytoin, Microsomes, Liver, Mixed Function Oxygenases, Polymorphism, Genetic, Steroid 16-alpha-Hydroxylase, Steroid Hydroxylases, Tolbutamide
Show Abstract · Added May 26, 2014
Genomic DNA was isolated from livers of 39 Japanese and 45 Caucasians and the genotypes of CYP2C9 and 2C19 genes were determined with PCR methods using synthetic oligonucleotide primers. Liver microsomes were also prepared from these human samples and activities for tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation were determined. The single base mutation of C416T (Arg144Cys) in CYP2C9 was detected in 22% of Caucasians but not in Japanese samples. Another single base mutation at A1061C (Ile359Leu) in the CYP2C9 gene was found with frequencies of about 8% in both races. We did not detect any individuals who have either homozygous Cys144/Cys144 or Leu359/Leu359 CYP2C9 variant nor both heterozygous Cys144-Ile359 and Arg144-Leu359 CYP2C9 variant in the human samples examined. The CYP2C19m2 genetic polymorphism was found only in Japanese people, while CYP2C19m1 type was determined in both races, with higher incidence in Japanese than in Caucasian population. Immunoblotting analysis of human liver microsomes suggested that CYP2C9 is a major component of the human CYP2C enzyme pool; it accounted for approximately 20% of total P450 in liver microsomes of both human populations. The levels of CYP2C19 protein were determined to be about 0.8% and 1.4% of total P450 (mean) in Japanese and Caucasians, respectively. We did not detect CYP2C19 protein in liver microsomes of humans who were genotyped for CYP2C19 gene as m1/m1, m1/m2, and m2/m2 variants but detected CYP2C9 protein in all of the samples examined. Good correlations were found between levels of CYP2C9 and activities of tolbutamide methyl hydroxylation (r = 0.77) and between levels of CYP2C19 and activities of S-mephenytoin 4'-hydroxylation (r = 0.86) in liver microsomes of the human samples examined. Tolbutamide methyl hydroxylation activities were lower in human samples with the Leu359 allele of CYP2C9 than those with the Cys144 allele and wild-type (Arg144-Ile359); the former type showed slightly higher K(m) values. When calculated on P450 basis, liver microsomes of individuals having m1/m1, m1/m2, and m2/m2 types of CYP2C19 had very low catalytic activities for S-mephenytoin 4'-hydroxylation. These results provide useful comparisons for pharmacokinetic and toxicokinetic models of some of the clinically used drugs that are oxidized by CYP2C proteins in humans.
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16 MeSH Terms
Expression of a human liver cytochrome P-450 protein with tolbutamide hydroxylase activity in Saccharomyces cerevisiae.
Brian WR, Srivastava PK, Umbenhauer DR, Lloyd RS, Guengerich FP
(1989) Biochemistry 28: 4993-9
MeSH Terms: Catalysis, Cytochrome P-450 Enzyme Inhibitors, Cytochrome P-450 Enzyme System, DNA, Electrophoresis, Gene Expression Regulation, Genetic Vectors, Hexobarbital, Humans, Liver, Mephenytoin, Mixed Function Oxygenases, Oxidation-Reduction, Plasmids, Saccharomyces cerevisiae, Spectrophotometry, Ultraviolet, Sulfaphenazole, Tolbutamide
Show Abstract · Added March 5, 2014
The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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18 MeSH Terms
Separation of human liver microsomal tolbutamide hydroxylase and (S)-mephenytoin 4'-hydroxylase cytochrome P-450 enzymes.
Srivastava PK, Yun CH, Beaune PH, Ged C, Guengerich FP
(1991) Mol Pharmacol 40: 69-79
MeSH Terms: Amino Acid Sequence, Aryl Hydrocarbon Hydroxylases, Chromatography, High Pressure Liquid, Cytochrome P-450 CYP2C19, Cytochrome P-450 Enzyme System, DNA, Humans, Mephenytoin, Microsomes, Liver, Mixed Function Oxygenases, Molecular Sequence Data, Saccharomyces cerevisiae, Sequence Homology, Nucleic Acid, Tolbutamide, Tritium
Show Abstract · Added March 5, 2014
Purification and immunoinhibition studies have suggested that the hydroxylations of (S)-mephenytoin and tolbutamide are catalyzed by rather similar forms of human liver cytochrome P-450 (P-450). However, the two activities are not well correlated in vivo; sulfaphenzaole is a selective inhibitor of tolbutamide hydroxylation, and expression of P-450 2C10 cDNA in yeast yields a protein that hydroxylates tolbutamide but not (S)-mephenytoin. The P-450 2C8, 2C9, and 2C10 cDNAs have all been isolated, and their sequences are known to be closely related (greater than 80%). Highly sensitive radiochromatographic assays were set up, and tolbutamide and (S)-mephenytoin hydroxylation activities were monitored during chromatography of human liver microsomal fractions. The two activities could be separated by chromatography, and proteins were purified to near-homogeneity that catalyzed either tolbutamide hydroxylation (P-450TB) or (S)-mephenytoin 4'-hydroxylation (P-450MP) but not both. Approximately 16 and 45% of the primary sequences of P-450TB and P-450MP, respectively, were determined by analysis of the tryptic peptides. The sequences of the P-450TB peptides matched those predicted by the P-450 2C9 and 2C10 cDNAs exactly; the P-450MP peptides showed two mismatches (of 219 residues) with the P-450 2C10 sequence. Proteins with the P-450 2C10 and P-450 2C9 sequences were expressed in Saccharomyces cerevisiae grown under different nutritional conditions, and both were found to be proficient in the hydroxylation of tolbutamide but not (S)-mephenytoin. We conclude, on the basis of this and previous work, that 1) P-450s 2C8, 2C9, and 2C10 all catalyze the hydroxylation of tolbutamide and 2) the protein involved in polymorphic (S)-mephenytoin 4'-hydroxylation is closely related to but distinct from P-450 2C8, 2C9, and 2C10.
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15 MeSH Terms