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L-type calcium channels play a critical role in maintaining lens transparency by regulating phosphorylation of aquaporin-0 and myosin light chain and expression of connexins.
Maddala R, Nagendran T, de Ridder GG, Schey KL, Rao PV
(2013) PLoS One 8: e64676
MeSH Terms: Animals, Aquaporins, Calcium Channel Blockers, Calcium Channels, L-Type, Connexins, Eye Proteins, Felodipine, Female, Gene Expression, Immunoblotting, Lens, Crystalline, Male, Mice, Mice, Inbred C57BL, Myosin Light Chains, Nifedipine, Phosphorylation, Reverse Transcriptase Polymerase Chain Reaction, beta-Crystallin B Chain
Show Abstract · Added May 27, 2014
Homeostasis of intracellular calcium is crucial for lens cytoarchitecture and transparency, however, the identity of specific channel proteins regulating calcium influx within the lens is not completely understood. Here we examined the expression and distribution profiles of L-type calcium channels (LTCCs) and explored their role in morphological integrity and transparency of the mouse lens, using cDNA microarray, RT-PCR, immunoblot, pharmacological inhibitors and immunofluorescence analyses. The results revealed that Ca (V) 1.2 and 1.3 channels are expressed and distributed in both the epithelium and cortical fiber cells in mouse lens. Inhibition of LTCCs with felodipine or nifedipine induces progressive cortical cataract formation with time, in association with decreased lens weight in ex-vivo mouse lenses. Histological analyses of felodipine treated lenses revealed extensive disorganization and swelling of cortical fiber cells resembling the phenotype reported for altered aquaporin-0 activity without detectable cytotoxic effects. Analysis of both soluble and membrane rich fractions from felodipine treated lenses by SDS-PAGE in conjunction with mass spectrometry and immunoblot analyses revealed decreases in β-B1-crystallin, Hsp-90, spectrin and filensin. Significantly, loss of transparency in the felodipine treated lenses was preceded by an increase in aquaporin-0 serine-235 phosphorylation and levels of connexin-50, together with decreases in myosin light chain phosphorylation and the levels of 14-3-3ε, a phosphoprotein-binding regulatory protein. Felodipine treatment led to a significant increase in gene expression of connexin-50 and 46 in the mouse lens. Additionally, felodipine inhibition of LTCCs in primary cultures of mouse lens epithelial cells resulted in decreased intracellular calcium, and decreased actin stress fibers and myosin light chain phosphorylation, without detectable cytotoxic response. Taken together, these observations reveal a crucial role for LTCCs in regulation of expression, activity and stability of aquaporin-0, connexins, cytoskeletal proteins, and the mechanical properties of lens, all of which have a vital role in maintaining lens function and cytoarchitecture.
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19 MeSH Terms
The bioelectrical basis and validity of gastrointestinal extracellular slow wave recordings.
Angeli TR, Du P, Paskaranandavadivel N, Janssen PW, Beyder A, Lentle RG, Bissett IP, Cheng LK, O'Grady G
(2013) J Physiol 591: 4567-79
MeSH Terms: Animals, Calcium Channel Blockers, Jejunum, Models, Biological, Myoelectric Complex, Migrating, Nifedipine, Stomach, Swine
Show Abstract · Added April 26, 2016
  Gastrointestinal extracellular recordings have been a core technique in motility research for a century. However, the bioelectrical basis of extracellular data has recently been challenged by claims that these techniques preferentially assay movement artifacts, cannot reproduce the underlying slow wave kinetics, and misrepresent the true slow wave frequency. These claims motivated this joint experimental-theoretical study, which aimed to define the sources and validity of extracellular potentials. In vivo extracellular recordings and video capture were performed in the porcine jejunum, before and after intra-arterial nifedipine administration. Gastric extracellular recordings were recorded simultaneously using conventional serosal contact and suction electrodes, and biphasic and monophasic extracellular potentials were simulated in a biophysical model. Contractions were abolished by nifedipine, but extracellular slow waves persisted, with unchanged amplitude, downstroke rate, velocity, and downstroke width (P>0.10 for all), at reduced frequency (24% lower; P=0.03). Simultaneous suction and conventional serosal extracellular recordings were identical in phase (frequency and activation-recovery interval), but varied in morphology (monophasic vs. biphasic; downstroke rate and amplitude: P<0.0001). Simulations demonstrated the field contribution of current flow to extracellular potential and quantified the effects of localised depolarisation due to suction pressure on extracellular potential morphology. In sum, these results demonstrate that gastrointestinal extracellular slow wave recordings cannot be explained by motion artifacts, and are of a bioelectrical origin that is highly consistent with the underlying biophysics of slow wave propagation. Motion suppression is shown to be unnecessary as a routine control in in vivo extracellular studies, supporting the validity of the extant gastrointestinal extracellular literature.
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8 MeSH Terms
Inhibition of human cytochrome P450 3A4 by cholesterol.
Shinkyo R, Guengerich FP
(2011) J Biol Chem 286: 18426-33
MeSH Terms: Anti-Arrhythmia Agents, Cells, Cultured, Cholesterol, Cytochrome P-450 CYP3A, Cytochrome P-450 CYP3A Inhibitors, Enzyme Inhibitors, Hepatocytes, Humans, Microsomes, Liver, Models, Biological, Nifedipine, Oxidation-Reduction, Quinidine, Vasodilator Agents
Show Abstract · Added March 7, 2014
If cholesterol is a substrate of P450 3A4, then it follows that it should also be an inhibitor, particularly in light of the high concentrations found in liver. Heme perturbation spectra indicated a K(d) value of 8 μM for the P450 3A4-cholesterol complex. Cholesterol inhibited the P450 3A4-catalyzed oxidations of nifedipine and quinidine, two prototypic substrates, in liver microsomes and a reconstituted enzyme system with K(i) ∼ 10 μM in an apparently non-competitive manner. The concentration of cholesterol could be elevated 4-6-fold in cultured human hepatocytes by incubation with cholesterol; the level of P450 3A4 and cell viability were not altered under the conditions used. Nifedipine oxidation was inhibited when the cholesterol level was increased. We conclude that cholesterol is both a substrate and an inhibitor of P450 3A4, and a model is presented to explain the kinetic behavior. We propose that the endogenous cholesterol in hepatocytes should be considered in models of prediction of metabolism of drugs and steroids, even in the absence of changes in the concentrations of free cholesterol.
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14 MeSH Terms
Chromatographic assays of drug oxidation by human cytochrome P450 3A4.
Sohl CD, Cheng Q, Guengerich FP
(2009) Nat Protoc 4: 1252-7
MeSH Terms: Chromatography, High Pressure Liquid, Cytochrome P-450 CYP3A, Humans, Kinetics, Mass Spectrometry, Microsomes, Liver, Nifedipine, Oxidation-Reduction, Testosterone
Show Abstract · Added March 26, 2014
Cytochrome P450 enzymes (P450s) are heme-thiolate mono-oxygenases involved in the oxidation of many endogenous and exogenous substrates. Herein, we describe two protocols for measuring the activity of a key enzyme of drug metabolism, P450 3A4. In this protocol, the substrate is incubated with human liver microsomes, the reaction is quenched, and the substrates and products are extracted and subjected to liquid chromatography (LC) separation and detection. Oxidation of the calcium-channel blocker nifedipine is measured using UV-Vis spectroscopy in-line with high performance liquid chromatography (HPLC). 6beta-Hydroxytestosterone formation from testosterone is measured by HPLC coupled to mass spectrometry (MS). Both of these procedures are rapid, requiring 2 h or less, and can be used to confirm and measure P450 3A4 activity and can also be used as a guide for developing other assays for measuring P450 catalysis. The separation strategy described here is more rapid than many available methods, except when ultra-performance liquid chromatography (UPLC) is used.
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9 MeSH Terms
Defective activity of recombinant cytochromes P450 3A4.2 and 3A4.16 in oxidation of midazolam, nifedipine, and testosterone.
Miyazaki M, Nakamura K, Fujita Y, Guengerich FP, Horiuchi R, Yamamoto K
(2008) Drug Metab Dispos 36: 2287-91
MeSH Terms: Alleles, Catalysis, Cytochrome P-450 CYP3A, Enzyme Activation, Humans, Isoenzymes, Microsomes, Liver, Midazolam, Nifedipine, Oxidation-Reduction, Recombinant Proteins, Substrate Specificity, Testosterone
Show Abstract · Added March 26, 2014
Cytochrome P4503A4 (CYP3A4) is the most abundant cytochrome P450 in adult human liver and small intestine and oxidizes numerous clinically, physiologically, and toxicologically important compounds. The metabolic activity of CYP3A4 in patients varies at least 10-fold in vivo, and CYP3A4 genetic variants are considered one of the causes of individual differences. The cDNAs for the CYP3A4(*)2 (S222P), (*)7 (G56D), (*)16 (T185S), and (*)18 (L293P) mutant alleles, found in high frequencies in Caucasians or Asians, were constructed by site-directed mutagenesis and expressed in an Escherichia coli expression system. Midazolam (MDZ), testosterone (TST), and nifedipine (NIF) were used to assess the catalytic activities of the CYP3A4 wild type (CYP3A4.1) and its variants. The catalytic activities of CYP3A4.2 and CYP3A4.16 were reduced (lower V(max) and increased K(m) relative to CYP3A4.1) for all substrates. The CYP3A4.7 showed lower V(max) values for MDZ and NIF (60 and 84%, respectively) and a higher K(m) (2-fold) for TST but not for MDZ or NIF. Although CYP3A4.18 showed low V(max) values for MDZ, NIF, and TST (88, 72, and 80% of CYP3A4.1, respectively), no significant differences were identified in the ratio V(max)(/K)(m). In summary, CYP3A4.2 and CYP3A4.16 exhibited significantly lower activity for MDZ, TST, and NIF oxidations than CYP3A4.1. Therefore, drugs metabolized by only CYP3A should be carefully administered to patients with these alleles.
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13 MeSH Terms
L-type Ca2+ channel blockers promote Ca2+ accumulation when dopamine receptors are activated in striatal neurons.
Eaton ME, Macías W, Youngs RM, Rajadhyaksha A, Dudman JT, Konradi C
(2004) Brain Res Mol Brain Res 131: 65-72
MeSH Terms: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine, Animals, Calcium, Calcium Channel Blockers, Calcium Channels, L-Type, Cells, Cultured, Corpus Striatum, Cyclic AMP Response Element-Binding Protein, Dizocilpine Maleate, Dopamine Antagonists, Drug Interactions, Excitatory Amino Acid Antagonists, Female, Neurons, Nifedipine, Oligonucleotides, Antisense, Phosphorylation, Pregnancy, Rats, Rats, Sprague-Dawley, Receptors, Dopamine D1, Receptors, N-Methyl-D-Aspartate
Show Abstract · Added May 27, 2014
Dopamine (DA) receptor-mediated signal transduction and gene expression play a central role in many brain disorders from schizophrenia to Parkinson's disease to addiction. While trying to evaluate the role of L-type Ca2+ channels in dopamine D1 receptor-mediated phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB), we found that activation of dopamine D1 receptors alters the properties of L-type Ca2+ channel inhibitors and turns them into facilitators of Ca2+ influx. In D1 receptor-stimulated neurons, L-type Ca2+ channel blockers promote cytosolic Ca2+ accumulation. This leads to the activation of a molecular signal transduction pathway and CREB phosphorylation. In the absence of dopamine receptor stimulation, L-type Ca2+ channel blockers inhibit CREB phosphorylation. The effect of dopamine on L-type Ca2+ channel blockers is dependent on protein kinase A (PKA), suggesting that protein phosphorylation plays a role in this phenomenon. Because of the adverse effect of activated dopamine receptors on L-type Ca2+ channel blocker action, the role of L-type Ca2+ channels in the dopamine D1 receptor signal transduction pathway cannot be assessed with pharmacological tools. However, with antisense technology, we demonstrate that L-type Ca2+ channels contribute to D1 receptor-mediated CREB phosphorylation. We conclude that the D1 receptor signal transduction pathway depends on L-type Ca2+ channels to mediate CREB phosphorylation.
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22 MeSH Terms
L-type voltage-gated Ca2+ channels modulate expression of smooth muscle differentiation marker genes via a rho kinase/myocardin/SRF-dependent mechanism.
Wamhoff BR, Bowles DK, McDonald OG, Sinha S, Somlyo AP, Somlyo AV, Owens GK
(2004) Circ Res 95: 406-14
MeSH Terms: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester, Actins, Animals, Aorta, Calcium Channel Blockers, Calcium Channels, L-Type, Cell Differentiation, Cells, Cultured, Gene Expression Profiling, Gene Expression Regulation, Developmental, Genes, fos, Intracellular Signaling Peptides and Proteins, Muscle, Smooth, Vascular, Myocytes, Smooth Muscle, Myosin Heavy Chains, Nifedipine, Nuclear Proteins, Organoids, Patch-Clamp Techniques, Potassium Chloride, Protein Transport, Protein-Serine-Threonine Kinases, RNA, Small Interfering, Rats, Reverse Transcriptase Polymerase Chain Reaction, Serum Response Element, Serum Response Factor, Trans-Activators, Transfection, rho-Associated Kinases, rhoA GTP-Binding Protein
Show Abstract · Added May 5, 2016
Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM alpha-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM alpha-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM alpha-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM alpha-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM alpha-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM alpha-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM alpha-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.
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31 MeSH Terms
Striatal proenkephalin gene induction: coordinated regulation by cyclic AMP and calcium pathways.
Konradi C, Macías W, Dudman JT, Carlson RR
(2003) Brain Res Mol Brain Res 115: 157-61
MeSH Terms: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine, Animals, Antihypertensive Agents, Calcium, Calcium Channel Agonists, Calcium Channel Blockers, Carcinogens, Cells, Cultured, Colforsin, Corpus Striatum, Cyclic AMP, Drug Interactions, Embryo, Mammalian, Enkephalins, Enzyme Inhibitors, Gene Expression Regulation, Developmental, Humans, Isoquinolines, Neurons, Nifedipine, Protein Precursors, Pyrroles, Rats, Rats, Sprague-Dawley, Sulfonamides, Tetradecanoylphorbol Acetate, Transcriptional Activation, Transfection
Show Abstract · Added May 27, 2014
Enkephalin modulates striatal function, thereby affecting motor performance and addictive behaviors. The proenkephalin gene is also used as a model to study cyclic AMP-mediated gene expression in striatal neurons. The second messenger pathway leading to proenkephalin expression demonstrates how cyclic AMP pathways are synchronized with depolarization. We show that cyclic AMP-mediated regulation of the proenkephalin gene is dependent on the activity of L-type Ca2+ channels. Inhibition of L-type Ca2+ channels blocks forskolin-mediated induction of proenkephalin. The Ca2+-activated kinase, Ca2+/calmodulin kinase, as well as the cyclic AMP-activated kinase, protein kinase A (PKA), are both necessary for the induction of the proenkephalin promoter. Similarly, both kinases are needed for the L-type Ca2+ channel-mediated induction of proenkephalin. This synchronization of second messenger pathways provides a coincidence mechanism that gates proenkephalin synthesis in striatal neurons, ensuring that levels are increased only in the presence of activated PKA and depolarization.
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28 MeSH Terms
Using full probability models to compute probabilities of actual interest to decision makers.
Harrell FE, Shih YC
(2001) Int J Technol Assess Health Care 17: 17-26
MeSH Terms: Arterial Occlusive Diseases, Bayes Theorem, Clinical Trials as Topic, Coronary Disease, Decision Making, Evidence-Based Medicine, Health Services Research, Humans, Models, Statistical, Nifedipine, Risk Assessment, Thrombolytic Therapy, Treatment Outcome, Vasodilator Agents
Show Abstract · Added February 28, 2014
The objective of this paper is to illustrate the advantages of the Bayesian approach in quantifying, presenting, and reporting scientific evidence and in assisting decision making. Three basic components in the Bayesian framework are the prior distribution, likelihood function, and posterior distribution. The prior distribution describes analysts' belief a priori, the likelihood function captures how data modify the prior knowledge; and the posterior distribution synthesizes both prior and likelihood information. The Bayesian approach treats the parameters of interest as random variables, uses the entire posterior distribution to quantify the evidence, and reports evidence in a "probabilistic" manner. Two clinical examples are used to demonstrate the value of the Bayesian approach to decision makers. Using either an uninformative or a skeptical prior distribution, these examples show that the Bayesian methods allow calculations of probabilities that are usually of more interest to decision makers, e.g., the probability that treatment A is similar to treatment B, the probability that treatment A is at least 5% better than treatment B, and the probability that treatment A is not within the "similarity region" of treatment B, etc. In addition, the Bayesian approach can deal with multiple endpoints more easily than the classic approach. For example, if decision makers wish to examine mortality and cost jointly, the Bayesian method can report the probability that a treatment achieves at least 2% mortality reduction and less than $20,000 increase in costs. In conclusion, probabilities computed from the Bayesian approach provide more relevant information to decision makers and are easier to interpret.
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14 MeSH Terms
Enhancement of cytochrome P-450 3A4 catalytic activities by cytochrome b(5) in bacterial membranes.
Yamazaki H, Nakajima M, Nakamura M, Asahi S, Shimada N, Gillam EM, Guengerich FP, Shimada T, Yokoi T
(1999) Drug Metab Dispos 27: 999-1004
MeSH Terms: Anesthetics, Intravenous, Bacteria, Calcium Channel Blockers, Cytochrome P-450 CYP3A, Cytochrome P-450 Enzyme System, Cytochromes b5, Escherichia coli, Humans, Hydroxylation, In Vitro Techniques, Kinetics, Membranes, Midazolam, Mixed Function Oxygenases, NADPH-Ferrihemoprotein Reductase, Nifedipine, Oxidation-Reduction, Recombinant Proteins, Testosterone
Show Abstract · Added May 26, 2014
Activities of testosterone, nifedipine, and midazolam oxidation by recombinant cytochrome P-450 (P-450) 3A4 coexpressed with human NADPH-P-450 reductase (NPR) in bacterial membranes (CYP3A4/NPR membranes) were determined in comparison with those of other recombinant systems and of human liver microsomes with high contents of CYP3A4. Growth conditions for Escherichia coli transformed with the bicistronic construct affected expression levels of CYP3A4 and NPR; an excess of NPR over P-450 in membrane preparations enhanced CYP3A4-dependent testosterone 6beta-hydroxylation activities of the CYP3A4/NPR membranes. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4/NPR membranes after addition to either bacterial membranes or purified enzymes. NPR was observed to enhance catalytic activity when added to the CYP3A4/NPR membranes, either in the form of bacterial membranes or as purified NPR (in combination with cholate and b(5)). Apparent maximal activities of testosterone 6beta-hydroxylation in CYP3A4/NPR membranes were obtained when the molar ratio of CYP3A4/NPR/b(5) was adjusted to 1:2:1 by mixing membranes containing each protein. Testosterone 6beta-hydroxylation, nifedipine oxidation, and midazolam 4- and 1'-hydroxylation activities in CYP3A4/NPR membranes plus b(5) systems were similar to those measured with microsomes of insect cells coexpressing CYP3A4 with NPR and/or of human liver microsomes, based on equivalent CYP3A4 contents. These results suggest that CYP3A4/NPR membrane systems containing b(5) are very useful models for prediction of the rates for liver microsomal CYP3A4-dependent drug oxidations.
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19 MeSH Terms