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PubMedDate not supplied

Pharmacological characterizations of recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes.

Recombinant human 5-HT1D alpha and 5-HT1D beta receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D alpha cell line) and Y-1 adrenocortical tumor cells (1D beta cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated cAMP accumulation (FSCA). [3H]LSD saturation studies indicated that 5-HT1D receptor expression levels were slightly higher in the 1D beta cell line (Bmax = 1334 +/- 134 fmol/mg protein) than in the 1D alpha cell line (Bmax = 900 +/- 218 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC50 approximately 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine > or = 5-carboxamidotryptamine (5-CT) > LSD > or = 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), with Ki/EC50 ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT1D alpha and 5-HT1D beta receptors with apparent dissociation constants (Kb values) of 12 +/- 1 nM and 3 +/- 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D alpha cell line, these compounds behaved as partial agonists in the 1D beta cell line. To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D beta cell line whereas nearly linear relationships were observed for these two compounds in the 1D alpha cell line. Moreover, KA/EC50 ratios of these compounds were significantly larger in the 1D beta (10-32) as compared to the 1D alpha (0.9-2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger-receptor reserve in the 1D beta cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists. Although the potencies (EC50 values) of compounds matched their respective affinity constants (Ki values) for the closely-related 5-HT1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT1D alpha receptor relative to the human 5-HT1D beta subtype may be responsible for the higher intrinsic activities observed in the 1D alpha cell line, even though receptor reserve is apparently lower in this system.

Adenylyl Cyclase Inhibitors↗
PubMed1998

Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring.

Cytochromes P450 (P450s or CYPs) constitute a superfamily of heme-thiolate proteins that play important roles in oxidative metabolism of endogenous and exogenous compounds. This review provides some limited history but addresses mainly the research progress on the cytochrome P450s in rainbow trout (Oncorhynchus mykiss), their purification, structures at the primary level, role in metabolism, responses to chemicals and environmental pollutants, application to biomonitoring and the effect of various factors on their expression or activities. Information obtained to date suggests that the rainbow trout P450 systems are as complex as those seen in mammals. Fourteen P450s have been purified from liver or trunk kidney to relatively high specific content. cDNAs belonging to seven different P450 families have been documented from trout liver, kidney and ovary. Two CYP1A genes, nine cDNAs containing open reading frames, and a cDNA fragment were entered into GenBank. Among them, CYP2K1, CYP2K3, CYP2K4, CYP2M1, CYP3A27 and CYP4T1 are the most recently described forms. CYP2K1, CYP2M1 and CYP4T1 represent newly identified P450 subfamilies first described in the rainbow trout. In many cases, the cloned rainbow trout P450s have subsequently been expressed in heterologous expressions systems such as COS-7 cells, yeast and baculovirus infected insect cells. Some of the overexpressed P450 isoforms have been partially characterized. Potential future research directions are discussed.

Animals↗
PubMed2003

Recognition molecule associated carbohydrate inhibits postsynaptic GABA(B) receptors: a mechanism for homeostatic regulation of GABA release in perisomatic synapses.

Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA(B) receptor-mediated activation of K(+) currents in CA1 pyramidal cells, which elevates extracellular K(+) concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA(B) receptors and inhibits GABA(B) receptor-activated K(+) currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA(A) receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA(B) receptor activity.

Animals↗
PubMed2005

Lack of functional D2 receptors prevents the effects of the D3-preferring agonist (+)-PD 128907 on dialysate dopamine levels.

Substantial pharmacological evidence is consistent with an inhibitory effect of D3 receptor activation on dopamine (DA) release. Although receptor selectivity of the ligands employed in initial studies has been questioned, studies employing new, more selective, compounds continue to support an involvement of this receptor subtype in regulating extracellular dopamine levels in the dorsal striatum and nucleus accumbens. Consistent with this hypothesis, microdialysis studies have shown that the dose-effect curve for (+)-PD 128907, a moderately selective D3 agonist, is shifted to the right in D3 knock out mice. The present microdialysis studies sought to further examine the role of D2 vs. D3 receptors in mediating (+)-PD 128907-evoked alterations in basal and depolarization-evoked DA levels. Dialysate DA levels were determined in D2 knock out mice and wild type littermate controls following both systemic and local administration of (+)-PD 128907. In view of regional differences in D3 receptor localization, studies were conducted in the nucleus accumbens, a D3 receptor rich area, and in the dorsal striatum, a region with low D3 receptor abundance. Systemic or reverse dialysis of (+)-PD 128907 into the nucleus accumbens significantly decreased basal and depolarization-evoked DA levels in wild type mice. A similar effect was observed in the dorsal striatum. Regardless of the route of administration, (+)-PD 128907 was ineffective in modulating DA levels in either brain region of D2 knock out mice. These data contrast with previous results in D3 knock out mice and indicate that the D2 receptor is necessary for the inhibition of presynaptic DA neurotransmission produced by a preferential D3 agonist. Based on the documented physical interaction of D2 and D3 receptors in heterologous expression systems, we put forth a hypothesis that reconciles the seemingly paradoxical results of this and previous microdialysis studies.

Analysis of Variance↗
PubMed1998

Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications.

Delta-aminolevulinic acid (ALA) is the precursor of porphyrin synthesis and has been recently used in vitro and in clinical studies as an endogenous photosensitizer for photodynamic therapy in the treatment of various tumors. For this purpose, ALA is given topically, systemically, or orally. When administered by the oral route, it shows excellent intestinal absorption. ALA is also efficiently reabsorbed in the renal proximal tubule after glomerular filtration. However, the pathways and mechanisms for its transmembrane transport into epithelial cells of intestine and kidney are unknown. Here we demonstrate that ALA uses the intestinal and renal apical peptide transporters for entering into epithelial cells. Kinetics and characteristics of ALA transport were determined in Xenopus laevis ooyctes and Pichia pastoris yeast cells expressing either the cloned intestinal peptide transporter PEPT1 or the renal form PEPT2. By using radiolabeled ALA and electrophysiological techniques in these heterologous expression systems, we established that: (a) PEPT1 and PEPT2 translocate 3H-ALA by saturable and pH-dependent transport mechanisms, (b) that ALA and di-/tripeptides, but not GABA or related amino acids, compete at the same substrate-binding site of the carriers, and (c) that ALA transport is electrogenic in nature as a consequence of H+/ALA cotransport. Reverse transcriptase-PCR analysis performed with specific primers for PEPT1 and PEPT2 in rabbit tissues demonstrates that, in particular, the PEPT2 mRNA is expressed in a variety of other tissues including lung, brain, and mammary gland, which have been shown to accumulate ALA. This suggests that these tissues could take up the porphyrin precusor via expressed peptide transporters, providing the endogenous photosensitizers for efficient photodynamic therapy.

Aminolevulinic Acid↗
PubMed1995

Heterologous expression of metabotropic glutamate receptors in adult rat sympathetic neurons: subtype-specific coupling to ion channels.

A novel heterologous expression system was used to examine the coupling of metabotropic glutamate receptors (mGluRs) to neuronal voltage-gated ion channels. Cytoplasmic injection of mGluR2 cRNA into adult rat sympathetic neurons resulted in the expression of receptors that negatively coupled to N-type Ca2+ channels through a pertussis toxin-sensitive pathway. Injection of mGluR1 alpha cRNA resulted in the expression of receptors that inhibited M-type K+ channels via a pertussis toxin-insensitive pathway. Coupling was restricted to specific transduction elements and effectors, since mGluR2 did not inhibit M channels and mGluR1 alpha had minimal effects on Ca2+ channels. These findings demonstrate that heterologously expressed, and thus unambiguously identified, mGluR subtypes modulate specific neuronal ion channels through discrete signal transduction pathways.

Animals↗
PubMed1992

Functional analysis of the promoter region of a nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L.

The 5'-flanking region of gln-gamma, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements using a series of 5' deletions and hybrid gln-gamma:: CaMV 35S promoters. The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems. In the first system, the chimaeric genes were transferred to Lotus corniculatus L. using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots. In the second system, the constructs were electroporated into tobacco mesophyll protoplasts. The results of the 5' deletion analysis showed that the sequence between -597 and -21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements. Sequences located between -2000 and -597 were able to stimulate expression in nodules but not protoplasts, while the region from -597 to -354 enhanced expression in both nodules and protoplasts. Results obtained with the hybrid gln-gamma::35S promoters showed that two overlapping restriction fragments (-516/-343 and -474/-293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner. Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between -516 and -446, and their possible role in regulating gln-gamma expression is discussed.

DNA Mutational Analysis↗
PubMed1993

Comparative study on the production of guar alpha-galactosidase by Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2 in continuous cultures.

Saccharomyces cerevisiae SU50B and Hansenula polymorpha 8/2, both carrying a multicopy integrated guar alpha-galactosidase, have been cultivated in continuous cultures, using various mixtures of carbon sources and cultivation conditions. Both S. cerevisiae SU50B and H. polymorpha 8/2 are stable and produce high levels of extracellular alpha-galactosidase in continuous cultures for more than 500 h. For these expression systems the strong inducible promoter systems GAL7 and methanol oxidase, respectively, were used. The induction of alpha-galactosidase synthesis by galactose in SU50B is limited by the low galactose uptake. Apart from that, at high dilution rates, the glucose repression is substantial, and a maximal expression level of 28.6 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1 could be obtained. In H. polymorpha, the induction of alpha-galactosidase synthesis, in addition to methanol oxidase synthesis using formaldehyde, is very effective up to 42 mg of extracellular alpha-galactosidase.g (dry weight) of biomass-1. Productivities in terms of specific production rate enable a good comparison with those of other heterologous expression systems in the literature. The productivities found with S. cerevisiae SU50B and H. polymorpha, 3.25 and 5.5 mg of alpha-galactosidase.g of biomass-1.liter-1.h-1, respectively, rank among the highest reported in the literature. Enzyme production and secretion in H. polymorpha are more complex. A two-peaked optimum is found in enzyme production. No clear explanation of this phenomenon can be given.

Alcohol Oxidoreductases↗
PubMed2000

Transport of N-acetylaspartate by the Na(+)-dependent high-affinity dicarboxylate transporter NaDC3 and its relevance to the expression of the transporter in the brain.

N-Acetylaspartate is a highly specific marker for neurons and is present at high concentrations in the central nervous system. It is not present at detectable levels anywhere else in the body other than brain. Glial cells express a high-affinity transporter for N-acetylaspartate, but the molecular identity of the transporter has not been established. The transport of N-acetylaspartate into glial cells is obligatory for its intracellular hydrolysis, a process intimately involved in myelination. N-Acetylaspartate is a dicarboxylate structurally related to succinate. We investigated in the present study the ability of NaDC3, a Na(+)-coupled high-affinity dicarboxylate transporter, to transport N-acetylaspartate. The cloned rat and human NaDC3s were found to transport N-acetylaspartate in a Na(+)-coupled manner in two different heterologous expression systems. The Michaelis-Menten constant for N-acetylaspartate was approximately 60 microM for rat NaDC3 and approximately 250 microM for human NaDC3. The transport process was electrogenic and the Na(+):N-acetylaspartate stoichiometry was 3:1. The functional expression of NaDC3 in the brain was demonstrated by in situ hybridization and reverse transcription-polymerase chain reaction as well as by isolation of a full-length functional NaDC3 from a rat brain cDNA library. In addition, the expression of a Na(+)-coupled high-affinity dicarboxylate transporter and the interaction of the transporter with N-acetylaspartate were demonstrable in rat primary astrocyte cultures. These studies establish NaDC3 as the transporter responsible for the Na(+)-coupled transport of N-acetylaspartate in the brain. This transporter is likely to be an essential component in the metabolic role of N-acetylaspartate in the process of myelination.

Animals↗
PubMed1996

Functional differences in Kv1.5 currents expressed in mammalian cell lines are due to the presence of endogenous Kv beta 2.1 subunits.

The voltage-sensitive currents observed following hKv1.5 alpha subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kv beta 2.1 subunit assembled with transfected hKv 1.5 protein. In contrast, both mRNA and protein analysis failed to detect a beta subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kv beta 2.1 expression. In the absence of Kv beta 2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were -0.2 +/- 2.0 and -9.6 +/- 1.8 mV, respectively. In the presence of Kv beta 2.1 these values were -14.1 +/- 1.8 and -22.1 +/- 3.7 mV, respectively. The beta subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kv beta 2.1 subunit can alter Kv1.5 alpha subunit function, 2) beta subunits are not required for alpha subunit expression, and 3) endogenous beta subunits are expressed in heterologous expression systems used to study K+ channel function.

Amino Acid Sequence↗
PubMed1989

High level gene expression in mammalian cells by a nuclear T7-phase RNA polymerase.

Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells. The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene. Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus. Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter. T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat. The unique properties of this heterologeous expression system are discussed.

Amino Acid Sequence↗
PubMed2006

Functional and molecular analysis of D-serine transport in retinal Müller cells.

D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.

Amino Acid Transport System ASC↗
PubMed1998

Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons.

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.

ATP-Binding Cassette Transporters↗
PubMed2004

Expression of human pyruvate carboxylase in insect cells using the baculovirus system.

Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of approximately 50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC.

Amino Acid Sequence↗
PubMed2000

Genetics and pathogenesis of malignant hyperthermia.

Malignant hyperthermia (MH) is a potentially life-threatening event in response to anesthetic triggering agents, with symptoms of sustained uncontrolled skeletal muscle calcium homeostasis resulting in organ and systemic failure. Susceptibility to MH, an autosomal dominant trait, may be associated with congenital myopathies, but in the majority of the cases, no clinical signs of disease are visible outside of anesthesia. For diagnosis, a functional test on skeletal muscle biopsy, the in vitro contracture test (IVCT), is performed. Over 50% of the families show linkage of the IVCT phenotype to the gene encoding the skeletal muscle ryanodine receptor and over 20 mutations therein have been described. At least five other loci have been defined implicating greater genetic heterogeneity than previously assumed, but so far only one further gene encoding the main subunit of the voltage-gated dihydropyridine receptor has a confirmed role in MH. As a result of extensive research on the mechanisms of excitation-contraction coupling and recent functional characterization of several disease-causing mutations in heterologous expression systems, much is known today about the molecular etiology of MH.

Animals↗
PubMed2006

A simple in vivo method for assessing changes of membrane-bound ion channel density in Xenopus oocytes.

Heterologous expression systems, such as Xenopus oocytes, are widely used to study the regulation and the structure function relationship of ion channels and transporters. In the case of ion channels, activity can be easily measured by conventional two-electrode voltage clamping. However, this method only measures the sum of the activity of all plasma membrane-bound channels. Therefore, this measurement cannot discriminate between effects on channel density and individual channel activity. To address this shortcoming, we have developed a simple assay to detect changes of membrane-bound channel density in intact oocytes. This nonradioactive assay relies on specific antibody binding in whole live cells utilizing a simple spectrophotometric measurement. This assay is linear over a wide range of channel expression levels and provides a simple cost-effective way of monitoring changes of membrane-bound channel density. Moreover, when the heterologous proteins poorly express at the plasma membrane, this method becomes advantageous to complex biochemical cell fractionation.

Animals↗
PubMed2004

Micro-electrode arrays in cardiac safety pharmacology: a novel tool to study QT interval prolongation.

Drug-induced QT interval prolongation is now a major concern in safety pharmacology. Regulatory authorities such as the US FDA and the European Medicines Agency require in vitro testing of all drug candidates against the potential risk for QT interval prolongation prior to clinical trials. Common in vitro methods include organ models (Langendorff heart), conventional electrophysiology on cardiac myocytes, and heterologous expression systems of human ether-a-go-go-related gene (hERG) channels. A novel approach is to study electrophysiological properties of cultured cardiac myocytes by micro-electrode arrays (MEA). This technology utilises multi channel recording from an array of embedded substrate-integrated extracellular electrodes using cardiac tissue from the ventricles of embryonic chickens. The detected field potentials allow a partial reconstruction of the shape and time course of the underlying action potential. In particular, the duration of action potentials of ventricular myocytes is closely related to the QT interval on an ECG. This novel technique was used to study reference substances with a reported QT interval prolonging effect. These substances were E4031, amiodarone, quinidine and sotalol. These substances show a significant prolongation of the field potential. However, verapamil, a typical 'false positive' when using the hERG assay does not cause any field potential prolongation using the MEA assay. Whereas the heterologous hERG assay limits cardiac repolarisation to just one channel, the MEA assay reflects the full range of mechanisms involved in cardiac action potential regulation. In summary, screening compounds in cardiac myocytes with the MEA technology against QT interval prolongation can overcome the problem of a single cell assay to potentially report 'false positives'.

Action Potentials↗
PubMed2001

Pitfalls in characterizing P450c17 mutations associated with isolated 17,20-lyase deficiency.

The cytochrome P450c17 enzyme system performs both the 17alpha-hydroxylase and 17,20-lyase reactions in the human adrenal glands and gonads. This 17,20-lyase activity is required for the biosynthesis of dehydroepiandrosterone, the C(19) precursor of sex steroids. Considerable evidence supports the idea that the 17,20-lyase activity of this system is particularly sensitive to alterations in the interactions between P450c17 and its cofactor proteins P450-oxidoreductase and cytochrome b(5). We have described two patients with the clinical phenotype of isolated 17,20-lyase deficiency in whom single amino acid replacement mutations in the redox partner binding site of P450c17 (R347H and R358Q) selectively ablate 17,20-lyase activity while preserving most 17alpha-hydroxylase activity. We have shown by computer modeling and detailed biochemical studies that mutations R347H and R358Q impair the interactions of P450c17 with P450-oxidoreductase and cytochrome b(5) (redox partners). Another mutation reported to cause isolated 17,20-lyase deficiency (F417C) does not map within the redox partner binding site, but might nonetheless alter the interaction of the mutant protein with redox partners. To study the interaction of the F417C mutation with P450 oxidoreductase and cytochrome b(5), we expressed the cDNA for this protein in yeast microsomes, a heterologous expression system in which the composition of redox partner proteins can be varied systematically. Although the full-length protein was expressed in quantities comparable to those of wild-type P450c17 in this system, the F417C mutation did not form a classical P450 difference spectrum and was devoid of both 17alpha-hydroxylase and 17,20-lyase activities. To ensure that this result was not unique to the yeast expression system, we also expressed wild-type P450c17 and the F417C mutation in COS-7 cells, and we again found that the F417C mutation was expressed, but was not active. To conclusively demonstrate that a particular mutation in P450c17 causes isolated 17,20-lyase deficiency, accurate enzymatic studies of the mutant protein must reproducibly show activities consistent with the diagnosis. Mutations R347H and R358Q are the only two such mutations found in humans proven to cause isolated 17,20-lyase deficiency.

Animals↗