ABSTRACT
Squalestatin1 (SQ1), a potent inhibitor of squalene synthase produced a dose-dependent induction of cytochromes P450 CYP2H1 and CYP3A37 mRNAs in chicken hepatoma cells. The effect of SQ1 was completely reversed by 25-hydroxycholesterol. Bile acids elicited an induction of CYP3A37 and CYP2H1 mRNA. Bile acids also reduced the phenobarbital induction of CYP2H1 but not of CYP3A37 mRNA. The effects of SQ1 and its reversal by 25-hydroxycholesterol and the effects of bile acids were reproduced in reporter gene assays with a phenobarbital-responsive enhancer unit of CYP2H1. These data suggest that an endogenous molecule related to cholesterol homeostasis regulates induction of drug-inducible CYPs.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cytochrome P-450 Enzyme System/genetics , Enzyme Inhibitors/pharmacology , Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors , Steroid Hydroxylases/genetics , Tricarboxylic Acids/pharmacology , Animals , Bile Acids and Salts/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/antagonists & inhibitors , Chickens , Cholesterol/metabolism , Cytochrome P-450 Enzyme System/biosynthesis , Dose-Response Relationship, Drug , Enhancer Elements, Genetic , Genes, Reporter , Hydroxycholesterols/pharmacology , Phenobarbital/pharmacology , Steroid Hydroxylases/biosynthesis , Transcriptional Activation , Tricarboxylic Acids/antagonists & inhibitors , Tumor Cells, CulturedABSTRACT
In a previous study we showed that activation of a presynaptically located metabotropic glutamate receptor (mGluR) with pharmacological properties of mGluR4a causes a facilitation of glutamate release in layer V of the rat entorhinal cortex (EC) in vitro. In the present study we have begun to investigate the intracellular coupling linking the receptor to transmitter release. We recorded spontaneous alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated excitatory postsynaptic currents (EPSCs) in the whole cell configuration of the patch-clamp technique, from visually identified neurons in layer V. Bath application of the protein kinase A (PKA) activator, forskolin, resulted in a marked facilitation of EPSC frequency, similar to that seen with the mGluR4a specific agonist, ACPT-1. Preincubation of slices with the PKA inhibitor H-89 abolished the effect of ACPT-1, as did preincubation with the adenylate cyclase inhibitor, SQ22536. Activation of protein kinase C (PKC) using phorbol 12 myristate 13-acetate (PMA) did not affect sEPSC frequency; however, it did abolish the facilitatory effect of ACPT-1 on glutamate release. A robust enhancement of EPSC frequency was seen in response to bath application of the specific PKC inhibitor, GF 109203X. Both H-89 and the group III mGluR antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) abolished the effects of GF 109203X. These data suggest that in layer V of the EC, presynaptic group III mGluRs facilitate release via a positive coupling to adenylate cyclase and subsequent activation of PKA. We have also demonstrated that the PKC system tonically depresses transmitter release onto layer V cells of the EC and that an interaction between mGluR4a, PKA, and PKC may exist at these synapses.
Subject(s)
Adenine/analogs & derivatives , Colforsin/analogs & derivatives , Cyclic AMP-Dependent Protein Kinases/physiology , Entorhinal Cortex/metabolism , Glutamic Acid/metabolism , Protein Kinase C/physiology , Receptors, Metabotropic Glutamate/physiology , Sulfonamides , Adenine/pharmacology , Animals , Colforsin/pharmacology , Cyclopentanes/antagonists & inhibitors , Cyclopentanes/pharmacology , Enzyme Activation/physiology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Glycine/analogs & derivatives , Glycine/pharmacology , In Vitro Techniques , Indoles/pharmacology , Isoquinolines/pharmacology , Male , Maleimides/pharmacology , Presynaptic Terminals/metabolism , Rats , Rats, Wistar , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Tricarboxylic Acids/antagonists & inhibitors , Tricarboxylic Acids/pharmacologyABSTRACT
The iron-molybdenum cofactor (FeMo-co) of nitrogenase is a Mo-Fe-S cluster that has been proposed as the site of substrate reduction for the nitrogenase enzyme complex. Biosynthesis of FeMo-co in Klebsiella pneumoniae requires at least six nif (nitrogen fixation) gene products. One of the nif genes, nifV, apparently encodes a homocitrate synthase. The synthesis and accumulation of homocitrate [(R)-2-hydroxy-1,2,4-butanetricarboxylic acid] in K.pneumoniae is correlated to the presence of a functional nifV gene. K.pneumoniae strains with mutations in nifV synthesize and accumulate an aberrant form of FeMo-co. Nitrogenase from NifV- mutants is capable of reducing some of the substrates of nitrogenase effectively (e.g. acetylene), but reduces N2 poorly. With the aid of an in vitro FeMo-co synthesis system, it recently has been established that homocitrate is an endogenous component of FeMo-co. Substitution of homocitrate with other carboxylic acids results in the formation of aberrant forms of FeMo-co with altered substrate reduction capability.
