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D F Covey

Publications and source records attributed to D F Covey.

At least 73 records · Page 4Linked to original sources
PubMed1994

Secosteroid mechanism-based inactivators and site-directed mutagenesis as probes for steroid hormone recognition by 3 alpha-hydroxysteroid dehydrogenase.

Rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) inactivates circulating androgens, progestins, and glucocorticoids. 3 alpha-HSD is a member of the aldo-keto reductase superfamily, and the X-ray structure of the apoenzyme shows the presence of an (alpha/beta)8 barrel [Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M., & Lewis, M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 2517-2521]. As yet, a three-dimensional structure of the ternary complex E.NADPH.steroid is unavailable. To identify regions of the enzyme involved in steroid hormone recognition, we have employed mechanism-based inactivators and site-directed mutagenesis. (3 RS)-1,10-Seco-5 alpha-estr-1-yne-3,17 beta-diol (1) and (17 RS)- 17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one (3) are secosteroids which contain latent Michael acceptors (alpha,beta-unsaturated alcohols) at opposite ends of the steroid nucleus (at the C-3 and C-17 positions, respectively). It was found that compounds 1 and 3 inactivated 3 alpha-HSD only in the presence of NAD+. The requirement for cofactor implies that 1 and 3 are oxidized to the corresponding alpha,beta-unsaturated ketones for inactivation to occur. Chemically prepared 17 beta-hydroxy-1,10-seco-5 alpha-estr-1-yn-3-one (2) and 14,15-secoandrost-4-en-15-yne-3,17-dione (4), the presumed products of 1 and 3 oxidation, behaved as stoichiometric inactivators of 3 alpha-HSD. In the presence and absence of NAD+, 2 and 4 inactivated > 50% of the enzyme in 10 s or less.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Alpha-spirocyclopentyl- and alpha-spirocyclopropyl-gamma-butyrolactones: conformationally constrained derivatives of anticonvulsant and convulsant alpha,alpha-disubstituted gamma-butyrolactones.

To further study the putative gamma-butyrolactone site of the GABAA/chloride channel complex, constrained derivatives of convulsant and anticonvulsant alpha,alpha-disubstituted gamma-butyrolactones (alpha-spirocyclopropyl- and alpha-spirocyclopentyl-gamma-butyrolactones) were synthesized and evaluated biologically. Most of the spirocyclopropyl agents were anticonvulsants when tested against pentylenetetrazole-induced seizures in mice. These agents effectively displaced 35[S]-tert-butylbicyclophosphorothionate (35[S]-TBPS), a ligand for the picrotoxin binding site of the GABAA/chloride channel, from rat neuronal membranes and affected the GABA-mediated current in hippocampal neurons. The monomethyl-substituted spirocyclopropyl agent with a methyl group cis to the carbonyl (15) potentiates GABA-induced current whereas the trans derivative (16) blocks the current. The only anticonvulsant in the spirocyclopentyl series was the unsubstituted spirocyclopentyl compound 2. All the other substituted spirocyclopentyl targets were inactive in vivo at the highest dose tested except for convulsant 9, which has a trans 2,5-dimethyl-substituted cyclopentyl ring. All the spirocyclopentyl derivatives displaced 35[S]-TBPS from rat neuronal membranes very effectively, and they also all potentiated GABA-induced chloride current except for convulsant 9 which blocked the current. From the data obtained in this investigation, it appears that when the volume occupied above and below the lactone ring is as large as that occupied by spirocyclopentyl agent 9, convulsant activity is observed. Groups with less volume in these areas either are inactive in the behavioral test or have anticonvulsant activity. When bound to the GABAA/chloride channel, the larger molecules may stabilize the closed state of the channel whereas the smaller molecules may stabilize the open state.

4-Butyrolactone↗
PubMed1994

Developmental alteration in GABAA receptor structure and physiological properties in cultured cerebellar granule neurons.

Although we now have extensive knowledge about the GABAA receptor subunits determining benzodiazepine modulation of channel function, little is known about subunits influencing other modulatory sites on the GABAA receptor-chloride channel complex. We have identified a developmental change in subunit composition of the GABAA receptor in cultured cerebellar granule neurons that eliminates benzodiazepine-mediated enhancement of GABA responses and alters modulation by a substituted gamma-butyrolactone. Based on data from sequential PCR experiments, we mimicked the functional properties of early and mature receptors with heterologous expression of specific subunit combinations. This report describes one of the most extensive cell- and site-specific developmental changes for an ion channel seen to date.

4-Butyrolactone↗
PubMed1993

Neurosteroid analogues: structure-activity studies of benz[e]indene modulators of GABAA receptor function. 1. The effect of 6-methyl substitution on the electrophysiological activity of 7-substituted benz[e]indene-3-carbonitriles.

The effect of 6-methyl substitution of the ability of 7-(2-hydroxyethyl)benz[e]indene-3-carbonitriles to potentiate GABA-mediated chloride current and to directly gate a chloride current in the absence of GABA in cultured rat hippocampal neurons was investigated. Structurally analogous steroid 17-carbonitriles that either contained or did not contain a 19-methyl group were also investigated. Compounds were evaluated at 1 microM for their ability to potentiate GABA-mediated currents and at 10 microM for current activation in the absence of GABA. The benz[e]indene 3(R)-carbonitriles and analogous steroid 17 alpha-carbonitriles had no effects in either assay. The benz[e]indene-3(S)-carbonitriles and analogous steroid 17 alpha-carbonitriles were active in both assays. Relative to the 6-unsubstituted benz[e]indene 3(S)-carbonitrile, the following effects of 6-methyl substituents were observed: a 6(a)-methyl group increased both activities; a 6(e)-methyl group decreased both activities; and 6,6-dimethyl substituents had opposing effects so that both activities remained similar to those of the 6-unsubstituted compound. The activities of the steroid 17 beta-carbonitriles were not affected significantly by the presence or absence of a 19-methyl group. A conformational analysis using molecular modeling methods was also performed for the benz[e]indene 3S-carbonitriles and the steroid 17 beta-carbonitriles. The ability of the different 6-methyl substituents to differentially effect the conformations of the flexible benz[e]indenes and the inability of the steroid 19-methyl group to alter the conformations of the rigid steroid 17 beta-carbonitriles are suggested to explain the results.

Androstanols↗
PubMed1993

Alkyl-substituted gamma-butyrolactones act at a distinct site allosterically linked to the TBPS/picrotoxinin site on the GABAA receptor complex.

Effects of alkyl-substituted gamma-butyrolactones and gamma-thiobutyrolactones on [35S]t-butylbicyclophosphorothionate (35S-TBPS) dissociation from the picrotoxinin receptor were studied. Unlike picrotoxinin, these lactones accelerated the dissociation rate of 35S-TBPS. Thus, previous reports that these lactones change the Kd but not the Bmax of 35S-TBPS in equilibrium binding experiments is explained not by competitive inhibition, but by an allosteric interaction with the 35S-TBPS binding site. These results indicate that modulatory effects of alkyl-substituted gamma-butyrolactones may result from their action at a distinct site on the gamma-aminobutyric acid (GABA)A receptor.

4-Butyrolactone↗
PubMed1993

Direct observation of a fluorinated anticonvulsant in brain tissue using 19F-NMR techniques.

A fluorinated derivative of an anticonvulsant gamma-butyrolactone [alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone; alpha-DFGBL] was synthesized as a probe for NMR spectroscopic observation of the drug in brain tissue. The fluorinated compound is an efficacious anticonvulsant in mice, and inhibits the specific binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to mouse brain membranes with a concentration dependence similar to that of the non-fluorinated compound alpha-ethyl-alpha-methyl-gamma-butyrolactone. Quantitative 19F-NMR spectroscopic studies, coupled with chromatographic measurements of drug tissue concentration, showed that virtually all of the alpha-DFGBL in brain was NMR-observable and that, following intraperitoneal injection, alpha-DFGBL rapidly achieved millimolar concentrations in brain. The 19F-NMR spectra of a alpha-DFGBL in brain and liver tissue were broad (1-2 ppm) and complex, exhibiting multiple chemical shift features. The major chemical shift features in these spectra were assigned on the basis of differential extraction and comparison of 19F spin-spin relaxation times (T2s) and 19F chemical shifts of alpha-DFGBL in tissue to those in pure solvents. The major feature at 10.4 ppm in the tissue spectra was assigned to a weakly polar, membrane-associated environment for the fluorinated compound, while the feature at 11.2 ppm was assigned to an aqueous environment for alpha-DFGBL. The drug was in slow exchange between these two environments in brain. In addition, the feature at lowest field (9.7-9.8 ppm) was identified as a water-soluble hydroxy-acid metabolite of alpha-DFGBL produced by the liver. These data indicate that gamma-butyrolactone anticonvulsants achieve high concentrations in brain, where they exist in several, largely membrane-associated, environments. These findings are consistent with the purported action of the gamma-butyrolactones as low-affinity modulators of gamma-aminobutyric acid-A channels.

4-Butyrolactone↗
PubMed1993

Inhibition of growth and appearance of estrogen-dependent rat mammary tumors by 10-propargylestr-4-ene-3,17-dione, an aromatase inhibitor.

The aromatase inhibitor 10-propargylestr-4-ene-3,17-dione (PED) has been evaluated in vivo as an anticancer agent. Prolonged administration of PED to rats bearing dimethylbenzanthracene-induced mammary tumors resulted in significant regression of hormone-responsive tumors within several days. Greater than 50% regression was generally observed after 14 days of treatment, irrespective of dose (1, 5, or 50 mg/kg body weight/day). In addition to tumor regression, a significantly increased incidence in tumor stasis was observed over the course of PED treatment. While all doses of PED examined were equipotent for both tumor regression and stasis, a dose-dependent inhibition of new tumor formation was observed in PED-treated rats. In control animals an average of 1.2 new tumors was observed during the experimental period; in contrast, averages of 0.5 tumors appeared in animals receiving 1 mg PED/kg body weight/day, 0.1 tumors at 5 mg/kg, and at 50 mg of PED/kg body weight/day, no new tumors occurred during the time PED was administered. The effects of PED on both regression of existing tumors and appearance of new tumors were reversed by co-administration of estradiol. Thus, PED impairs estrogen-dependent mammary tumor growth, resulting in cessation of new growth and regression of responsive tumors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1993

Multiple mechanisms of picrotoxin block of GABA-induced currents in rat hippocampal neurons.

1. We have examined the effect of picrotoxin on GABA-induced currents in dissociated rat hippocampal neurons. In addition, we used the putative picrotoxin receptor antagonist, alpha-isopropyl-alpha-methyl-gamma-butyrolactone (alpha IMGBL), and the picrotoxin agonist, beta-ethyl-beta-methyl-gamma-butyrolactone (beta EMGBL) to explore the mechanisms of picrotoxin's interaction with the GABA-Cl- receptor-ionophore complex. 2. The picrotoxin block of GABA current was use dependent, suggesting that the site of picrotoxin block is exposed by the conformational change initiated by GABA binding to the receptor. 3. The alkyl-substituted butyrolactone antagonist, alpha IMGBL, selectively blocked the use-dependent mechanism of picrotoxin effect. After the apparent complete inhibition of the use-dependent effect, there was a residual picrotoxin effect that was independent of the time or concentration of GABA application. This indicates that the picrotoxin block of the GABA current is mediated by two different mechanisms. alpha IMGBL influences just one of these mechanisms. 4. The picrotoxin receptor agonist, beta EMGBL, exclusively blocked the GABA current in a use-dependent manner. Consistent with a use-dependent mechanism, the rate of onset of block increased with GABA concentration. Surprisingly, the fraction of GABA current block decreased with increasing GABA concentration. 5. These results suggest that the relationship of picrotoxin and gamma-butyrolactones with the GABA-Cl- receptor-ionophore is quite complex. They are consistent with at least two possible models of agonist-antagonist interactions. Both cases require different antagonist affinities for the various kinetic states of the GABA-Cl- receptor-ionophore. However, there is no need to require that either picrotoxin or beta EMGBL acts as an open channel blocker.

4-Butyrolactone↗
PubMed1992

Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.

3 beta-Hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase copurify from human placental microsomes as a single enzyme protein. The affinity-alkylating secosteroid, 5,10-secoestr-4-yne-3,10,17-trione, inactivates the dehydrogenase and isomerase reactions in a time-dependent manner, but which of the two activities is targeted depends on the concentration of secosteroid. At 2-5 microM secosteroid, the dehydrogenase activity is alkylated in a site-specific manner (pregnenolone slows inactivation) that follows first-order inactivation kinetics (KI = 4.2 microM, k3 = 1.31 x 10(-2) min-1). As the secosteroid level increases from 11 to 30 microM, dehydrogenase is paradoxically inactivated at progressively slower rates, and pregnenolone no longer protects against the alkylator. The inactivation of isomerase exhibits the expected first-order kinetics (KI = 31.3 microM, k3 = 6.42 x 10(-2) min-1) at 11-30 microM secosteroid. 5-Androstene-3,17-dione protects isomerase from inactivation by 15 microM secosteroid, but the substrate steroid unexpectedly fails to slow the inactivation of isomerase by a lower concentration of alkylator (5 microM). A shift from a dehydrogenase to an isomerase conformation in response to rising secosteroid levels explains these results. Analysis of the ligand-induced conformational change along with cofactor protection data suggests that the enzyme expresses both activities at a bifunctional catalytic site. According to this model, the protein begins the reaction sequence as 3 beta-hydroxysteroid dehydrogenase. The products of the first step (principally NADH) promote a change in protein conformation that triggers the isomerase reaction.

3-Hydroxysteroid Dehydrogenases↗
PubMed1992

Warfarin sodium. Practitioner beware.

The use of warfarin sodium in the US has increased dramatically over the last 40 years. Warfarin sodium or Coumadin is the most popular oral anticoagulant used in the US. The podiatric physician frequently encounters patients taking this agent and therefore needs to understand its potential impact. This article will familiarize the reader with the history, pharmacology, clinical applications, side effects, and frequent drug interactions encountered with warfarin sodium.

Contraindications↗
PubMed1992

Effects of a benz[e]indene on gamma-aminobutyric acid-gated chloride currents in cultured postnatal rat hippocampal neurons.

Benz[e]indenes (BIs) are tricyclic molecules that can be envisioned as steroids without an A-ring. Because certain steroids are known to alter gamma-aminobutyric acid (GABA) responses in central neurons, we examined the effects of a substituted BI resembling 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) on GABA-gated chloride currents in cultured postnatal rat hippocampal neurons. The compound, BI-1, reversibly potentiated GABA currents at concentrations of > 10 nM, with an EC50 value of 0.2 microM. BI-1 increased the apparent affinity of GABA for its receptor, decreasing the GABA EC50 from 9 microM to 3 microM. BI-1 had no effect on the shape of the GABA current-voltage relationship and did not alter the GABA reversal potential. The effects of BI-1 were not altered by benzodiazepine or picrotoxin site antagonists. At concentrations up to 10 microM, where maximal effects on GABA currents were seen, BI-1 did not directly activate a membrane current. This contrasts with the effects of 3 alpha-OH-DHP, which activated chloride currents at concentrations that were subsaturating for GABA potentiation. These results suggest that the BIs may be useful for determining the mechanisms by which steroids potentiate GABA responses and directly gate chloride channels.

Animals↗
PubMed1991

Synthesis and structure-activity studies of alkyl-substituted gamma-butyrolactones and gamma-thiobutyrolactones: ligands for the picrotoxin receptor.

A series of gamma-butyrolactones and gamma-thiobutyrolactones possessing a variety of alkyl groups and alkyl-substitution patterns was prepared and evaluated for anticonvulsant and convulsant activity. Behavioral studies performed on these compounds suggest that maximal anticonvulsant activity (against maximal electroshock and pentylenetetrazol) results when three or four carbon atoms are present at the alpha-position. For convulsant potency, a similar dependence on the size of the alkyl chain at the beta-position was observed. Additional gamma-dimethyl groups were found to increase the convulsant potency of a beta-substituted compound and to cause an alpha-substituted anticonvulsant to become a convulsant. In general, sulfur for oxygen heteroatom substitution in the alpha-substituted lactones resulted in improved anticonvulsant potency and spectrum of activity. Binding of these compounds to the picrotoxin site of the GABA receptor complex was demonstrated with a [35S]-tert-butylbicyclophosphorothionate radioligand binding assay. Measurements of brain concentrations for selected compounds supports a hypothesis that correlates binding to the picrotoxin site with the pharmacological effects of these compounds.

4-Butyrolactone↗
PubMed1991

Gamma-butyrolactone antagonism of the picrotoxin receptor: comparison of a pure antagonist and a mixed antagonist/inverse agonist.

Multiple receptors modulate the ion channel gated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). gamma-Butyrolactones and gamma-thiobutyrolactones are compounds that act at the picrotoxin recognition site on the GABA receptor complex as either agonists or inverse agonists, depending on the nature of the alkyl substituents. Here we have compared the effects of two gamma-butyrolactones, alpha-ethyl-alpha-methyl-gamma-butyrolactone (alpha EMGBL) and alpha-isopropyl-alpha-methyl-gamma-butryolactone (alpha IMGBL), on GABA currents and inhibitory postsynaptic currents (IPSCs) in cultured, voltage-clamped, rat hippocampal neurons. alpha EMGBL also decreased the rate of IPSC decay without altering IPSC peak amplitude. At higher GABA concentrations (30 microM), alpha EMGBL has already been shown to block picrotoxin receptor agonists and inverse agonists. Thus, alpha EMGBL is a mixed antagonist/inverse agonist. In contrast to alpha EMGBL, alpha IMGBL had no effect on responses to either 0.5 or 30 microM GABA or on IPSCs, but it was able to block the effects of picrotoxin receptor agonists and inverse agonists. Therefore, alpha IMGBL is the first pure antagonist to be described for the picrotoxin receptor. The main conductance state of the GABA-gated channel probably has two or more open states, brief openings associated with binding of a single GABA molecule and longer openings due to the binding of two GABA molecules. We were able to simulate the results obtained with alpha EMGBL, using a computer model, by assuming that alpha EMGBL altered only the opening and closing rate constants for the monoliganded open channel of the GABA receptor. In addition to having site-selective actions, these results suggest that drugs modulating the GABA-linked chloride ionophore may be specific for the kinetic state of the GABA-gated channel.

4-Butyrolactone↗
PubMed1990

Physiological modulation of the GABA receptor by convulsant and anticonvulsant barbiturates in cultured rat hippocampal neurons.

The actions of convulsant and sedative barbiturates on responses to gamma-aminobutyric acid (GABA) application and on inhibitory postsynaptic currents were compared using voltage-clamp techniques in cultured rat hippocampal neurons. The convulsant barbiturates, 5-ethyl-5-(3-methylbut-2-enyl) barbituric acid (3M2B), and (+)-5-ethyl-5-(1,3-dimethylbutyl) barbituric acid [+)-DMBB), and the sedative barbiturate, 5-ethyl-5-(3-methylbutyl) barbituric acid (3MB), all potentiated GABA-mediated chloride currents. In addition, these compounds prolonged the duration of GABAergic inhibitory postsynaptic currents. The similarity between the action of convulsant and sedative barbiturates suggests that the convulsant activity of 3M2B and (+)-DMBB are not mediated by their actions at GABAergic synapses.

Animals↗
PubMed1990

The effects of in vivo administration of 10-propargylestr-4-ene-3,17-dione on rat ovarian aromatase and estrogen levels.

We have previously demonstrated that 10-propargylestr-4-ene-3,17-dione (PED) functioned as an irreversible inhibitor of rat ovarian aromatase in vitro. These studies were undertaken to examine the in vivo effects of PED on rat ovarian aromatase activity and estrogen production. In the current experiments, a single injection of PED (0.5 or 2.5 mg/kg) was found to maximally inhibit aromatase at 3 h regardless of dose. Significant inhibition of enzyme activity by PED was observed beyond 18 h, although some recovery was noted at the lower dose (0.5 mg/kg). Concomitantly, ovarian estrogen levels were also maximally reduced at 3 h, however ovarian estrogen levels returned toward control values prior to the recovery in enzyme activity. Even though significant inhibition of enzyme activity was observed at 12 h following a single injection of PED, the effect of double injections of the inhibitor at 12 h intervals was surprisingly not cumulative. Similarly, continued multiple injections of PED revealed significant inhibition of enzyme activity and estrogen production several hours after the injection, but variations in effectiveness were observed by 12 h which changed in accordance with a circannual cycle in aromatase. Apparently other factors are involved with maintaining aromatase levels and compensating for reduced enzyme activity. These mechanisms are evidenced by a continuation of the rat reproductive cycle with prolonged PED administration and a reduced influence of PED in regard to enzyme inhibition at certain times of the year. Despite these variations in the duration of action of PED, no comparable changes were observed in effectiveness as an anti-tumor agent. These results suggest that complex mechanisms exist which regulate the activity of aromatase in order to maintain estrogen production. Further research using compounds such as PED may assist in elucidating the factors that modulate ovarian estrogen production.

Androstenedione↗
PubMed1990

Modulation of the picrotoxin receptor by fluorinated ethyl, methyl-butyrolactones.

A number of investigators have shown that gamma-butyrolactones bind to the picrotoxin site on the gamma-aminobutyric acid (GABA) receptor. We examined the effects of three fluorinated gamma-butyrolactone derivatives on GABA currents and synaptic currents in cultured hippocampal neurons. alpha-(2,2,2-trifluoroethyl)-alpha-methyl-gamma-butyrolactone was a partial picrotoxin agonist, meaning that like picrotoxin, it blocked GABA currents, but not completely. This action is in marked contrast to that of its parent compound, alpha-ethyl-alpha-methyl-gamma-butyrolactone, which is a mixed inverse agonist/antagonist, enhancing GABA currents at low GABA concentration (less than or equal to 1 microM) and antagonizing picrotoxin block of GABA currents at higher GABA concentrations (approximately 30 microM). The properties of two difluorinated derivatives were found to be very different from the trifluorinated compound. Both alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone and alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-thiobutyrolactone+ ++ dramatically increased the current produced by low concentrations of GABA. The former had less marked inverse agonist effects at higher GABA concentrations but behaved instead like a picrotoxin antagonist. In addition, alpha-(1,1-difluoroethyl)-alpha-methyl-gamma-butyrolactone reduced both excitatory and inhibitory synaptic currents suggesting a separate effect on presynaptic transmitter release through an unknown mechanism. These experiments support the hypothesis that gamma-butyrolactones can have a variety of effects at the picrotoxin receptor and indicate that specific properties of these compounds can be altered drastically by fluorination.

4-Butyrolactone↗