Showing papers in "Molecular Pharmacology in 1984"

The kinetics of competitive radioligand binding predicted by the law of mass action.

Abstract: Although equilibrium competitive radioligand binding studies are often used to characterize hormone and neurotransmitter receptors, the kinetics of such experiments have not been extensively explored. The interactions of the radioligand and competitor with the receptors can be described by two differential equations which can be solved to yield a single equation describing the binding of the radioligand as a function of time. This equation has several applications: First, it can be used to simulate competitive binding reactions under defined conditions. Second, fitting experimental data to this equation allows one to determine the association and dissociation rate constants of the competing ligand, parameters that cannot be derived from equilibrium experiments. Furthermore, this method can be used to determine the KI of the competing drug from data acquired before equilibrium is reached. Third, mathematical analysis of the binding equation allowed us to answer two specific questions regarding the kinetics of competitive radioligand binding: how long such an incubation takes to equilibrate, and how the IC50 varies over time. The answers to these questions depended, to a large extent, on the relative values of the dissociation rate constants of the radioligand and competitor, which can be determined as noted above. When the competitor dissociates from the receptors more rapidly than the radioligand, the IC50 first decreases and then increases, but never has a value less than the KI. At low radioligand concentrations, equilibrium is reached in the same amount of time required of the radioligand to dissociate completely from the receptors as determined in an "off-rate experiment." At higher concentrations of radioligand this time is halved. When the competitor dissociates from the receptor more slowly than does the radioligand, then the time required to equilibrate depends only on the dissociation rate constant of the competitor, and the IC50 decreases over time.

Cannabinoid inhibition of adenylate cyclase. Pharmacology of the response in neuroblastoma cell membranes.

Abstract: Adenylate cyclase in plasma membranes was inhibited by micromolar concentrations of delta 8-tetrahydrocannabinol and delta 9-tetrahydrocannabinol and by levonantradol and desacetyllevonantradol. This inhibition was noncompetitive for stimulation of the enzyme at the prostanoid receptor by prostaglandin E1 or prostacyclin, or at the peptide receptor by secretin or vasoactive intestinal peptide. Forskolin-activated adenylate cyclase was also inhibited by cannabimimetic agents. Inhibition by cannabinoid compounds was neither synergistic nor additive with muscarinic or alpha-adrenergic agents when each was present at maximally inhibitory concentrations. Cannabinoid inhibition was not blocked by atropine, yohimbine, or naloxone, suggesting that muscarinic, alpha 2-adrenergic and certain opiate receptors may not be required for the response. The inhibition of adenylate cyclase was specific for psychoactive cannabinoids, since cannabinol and cannabidiol produced minimal or no response. Inhibition was also stereoselective, since dextronantradol did not produce the response. A biphasic log dose-response curve was observed for each of the cannabinoid drugs, such that reversal of the inhibition occurred at 3-10 microM. Possible mechanisms for the effects of cannabinoid drugs on adenylate cyclase activity are discussed.

Activation of calmodulin by various metal cations as a function of ionic radius.

Abstract: The active form of calmodulin is a Ca2+ . calmodulin complex. The purpose of this investigation was to determine whether other metal cations substitute for Ca2+ to activate calmodulin. Binding of Ca2+ resulted in an altered conformation of calmodulin with an increased quantum yield in its tyrosine fluorescence. Qualitatively similar results were obtained with Zn2+, Mn2+, Cd2+, Hg2+, Sr2+, Pb2+, Tb3+, Sm3+, and La3+. The relative extents of fluorescence enhancement by these cations were related to their ionic radii: all cations with ionic radii close to Ca2+ (0.99 A) increased tyrosine fluorescence, whereas those with different ionic radii were not effective, or much less so. The change in calmodulin conformation by the cations was confirmed by its altered electrophoretic mobility on polyacrylamide gels. Cations that change the conformation of calmodulin allow it to stimulate phosphodiesterase. The relative extents of stimulation of phosphodiesterase by cations were also related to their ionic radii. Finally, the ability of metal cations to inhibit Ca2+ binding was similarly related to their ionic radii. In general, the closer the radius of a metal cation was to that of Ca2+, the more effective was the cation to substitute for Ca2+. The range of effective ionic radii was approximately 1 +/- 0.2 A. Calmodulin-stimulated phosphodiesterase activity by the cations was reversed by trifluoperazine, an antagonist of calmodulin.

[3H]mazindol binding associated with neuronal dopamine and norepinephrine uptake sites.

Abstract: [3H]Mazindol labels neuronal dopamine uptake sites in corpus striatum membranes (KD = 18 nM) and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes (KD = 4 nM). The potencies of various inhibitors of biogenic amine uptake in reducing [3H]mazindol binding in striatal membranes correlate with their potencies for inhibition of neuronal [3H]dopamine accumulation, whereas their potencies in reducing [3H]mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal [3H]norepinephrine accumulation. Similar to the dopamine and norepinephrine uptake systems, [3H]mazindol binding in all three tissues is dependent upon sodium (with potassium, lithium, rubidium, and Tris being ineffective substitutes) and chloride (with sulfate and phosphate being ineffective substitutes). In membranes of the cerebral cortex and salivary gland, half-maximal stimulation is observed at 50-80 mM NaCl, whereas in membranes of the corpus striatum half-maximal stimulation occurs at 240 mM NaCl. In striatal membranes NaCl increases the affinity of [3H]mazindol binding with no effect on the maximal number of sites. The enhancement of affinity is due to a selective slowing of the dissociation of the ligand from its binding site. The association of [3H]mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of [3H]mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, the association of [3H]mazindol binding sites with neuronal norepinephrine uptake sites in cerebral cortex is supported by the reduction of [3H]mazindol binding to cortical membranes following destruction of noradrenergic neurons by N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine.

gamma-aminobutyric acid B receptors are negatively coupled to adenylate cyclase in brain, and in the cerebellum these receptors may be associated with granule cells.

Abstract: Baclofen and gamma-aminobutyric acid (GABA) are shown to inhibit basal adenylate cyclase activity in brain of rat. The response is mediated through the GABAB receptor, and the rank order of potency for agonists is (-)-baclofen (EC50 = 4 microM) greater than GABA (EC50 = 17 microM) greater than muscimol greater than (+)-baclofen. GABAA agonists are not effective inhibitors of cyclase activity. The response is bicuculline-insensitive, and diazepam does not modify the GABA or (-)-baclofen inhibition of adenylate cyclase. Studies with neurologically mutant mice correlated a loss in GABAB receptor-mediated inhibition of cyclase with a loss in cerebellar granule cells. Thus, the GABAB receptor is negatively coupled to adenylate cyclase in various brain areas, and, in the cerebellum, data suggest a granule cell localization of this activity.

Structure-activity relationships of amiloride and certain of its analogues in relation to the blockade of the Na+/H+ exchange system.

Abstract: Amiloride and 38 amiloride analogues were tested for their inhibitory action on the Na+/H+ exchanger of chick skeletal muscle cells. The unsubstituted guanidino group of amiloride is essential for the activity of the molecule, since substitution of its results in almost inactive molecules. Selected modification of position 3 and 5 substituents of amiloride have a less dramatic effect on its potency. Substitution of the 5-amino group of amiloride with alkyl or alkenyl groups produced compounds that were up to 140 times more potent than amiloride in inhibiting the Na+/H+ exchanger. Such molecules would appear to be preferable to use in place of amiloride in biochemical and physiological studies of the Na+/H+ exchanger.

Site-directed alkylation of multiple opioid receptors. I. Binding selectivity.

Abstract: We report a method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites. We used radioligands that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands, sufentanil for mu sites, [D-Ala2, D-Leu5] enkephalin for delta sites, and dynorphin A for kappa sites. After enrichment for mu receptors, [3H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [3H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but we found no evidence for residual mu or delta sites in kappa-enriched membranes. We used this method to identify ligands that are highly selective for each of the three types of sites: Tyr-D-Ala-Gly-(Me)Phe-Gly-ol, sufentanil, and morphiceptin for mu sites; (D- Pen2 , D- Pen5 ]enkephalin and [D- Pen2 ,L- Pen5 ]enkephalin for delta sites; and tifluadom and U50 ,488 for kappa sites.

Inhibition of binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels by local anesthetics.

Abstract: The effects of several local anesthetics on the binding of ligands to receptors associated with voltage-sensitive sodium channels in rat brain synaptosomes have been examined. In the presence of 0.3 microM scorpion toxin, the 13 local anesthetics tested inhibited the specific binding of [3H]batrachotoxinin A 20 alpha-benzoate [( 3H]BTX-B), a ligand which binds to a receptor site responsible for the activation of sodium channel ion flux, in a dose-dependent fashion, with KD values ranging from 1.2 microM for tetracaine to 1.58 mM for benzocaine. A plot of log KD from these binding experiments against log K0.5 for inhibition of sodium currents by local anesthetics from electrophysiological experiments yielded a regression line with a slope of 0.84 and a correlation coefficient, r, of 0.86, demonstrating that the inhibition of [3H]BTX-B binding by local anesthetics occurs within a concentration range of physiological relevance. Tetracaine had little effect on basal 125I-labeled scorpion toxin binding to synaptosomes in the absence of batrachotoxin. However, in the presence of batrachotoxin, tetracaine inhibited the batrachotoxin-dependent increase in scorpion toxin binding (KD = 2.0 microM) in a dose-dependent manner, suggesting that inhibition of [3H]BTX-B binding by local anesthetics does not occur through binding at the scorpion toxin binding site. The inhibition of [3H]BTX-B binding by lidocaine was reversible within 30 min when samples were diluted from 10(-3)M to 10(-4) M lidocaine. Scatchard analysis of [3H]BTX-B binding to synaptosomes showed that bupivacaine and tetracaine reduced receptor affinity without decreasing maximal binding capacity. This reduction in receptor affinity in the presence of local anesthetics appears to be due, at least in part, to an increased rate of ligand dissociation from the receptor-ligand complex, suggesting an indirect allosteric mechanism for the inhibition of [3H]BTX-B binding by local anesthetics. Analysis of the effects of local anesthetics in terms of an allosteric model of drug action showed that they bind to inactive states of sodium channels with at least a 10-fold higher affinity than active states. A 7-fold difference in KD for inhibition of [3H]BTX-B binding between the local anesthetic stereoisomers RAC 109 I and RAC 109 II was observed. Similarly, the dissociation rate constant for the [3H]BTX-B/receptor complex was increased 9.3-fold in the presence of RAC 109 II and 4.3-fold in the presence of a comparable concentration of RAC 109 I.(ABSTRACT TRUNCATED AT 250 WORDS)

Carboxyl-terminal tripeptidyl hydrolysis of substance P by purified rabbit lung angiotensin-converting enzyme and the potentiation of substance P activity in vivo by captopril and MK-422.

Abstract: The hydrolysis of substance P is catalyzed by purified rabbit lung angiotensin-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1). The kcat/Km for the reaction at 37 degrees is 3.3 +/- 0.3 X 10(3) M-1 sec-1, which is 60 times less than that which has been reported for the hydrolysis of angiotensin I. The initial site of hydrolysis is the antipenultimate peptide bond, which generates the tripeptide amide (Gly-Leu-Met-NH2). This hydrolysis is inhibited by the angiotensin-converting enzyme inhibitors captopril, MK-422, and EDTA, and is dependent on the concentration of chloride ion. Both captopril and MK-422 potentiate the substance P-induced stimulation of salivation in rats. Thus, angiotensin-converting enzyme may be one of the enzymes that degrade substance P in vivo.

Opiate-receptor interactions on single locus coeruleus neurones.

Abstract: Intracellular recordings were made from neurones of the rat locus coeruleus (LC) which were located in a slice of pons superfused in vitro. Opioid agonists and antagonists were applied by adding them to the superfusing solution; normorphine and enkephalin analogues were also applied by ejecting a few nanoliters of a solution which contained the drugs from a pipette situated above the tissue slice. Opioid agonists hyperpolarized LC neurones. This has been shown previously to result from an increase in the membrane potassium conductance. The lowest concentration of normorphine which was effective was 30 nM, the EC50 was 1 microM, and the maximum effect was observed with 30 microM. The irreversible antagonist beta-funaltrexamine (beta-FNA) was used to estimate the dissociation equilibrium constants; these ranged from 9-16 microM for normorphine and [Met5]enkephalin and was about 2 microM for [D-Ala2,D-Leu5]enkephalin. beta-FNA also blocked the hyperpolarization caused by [D-Ala2,D-Leu5]enkephalin, ethylketacyclazocine, and [D-Ser2,D-Leu5] enkephalin-Thr. Naloxone reversibly antagonized the hyperpolarizations caused by normorphine and [D-Ala2,D-Leu5]enkephalin, with a dissociation equilibrium constant of 2 nM. It is suggested that the opioid hyperpolarization of LC neurones is mediated by a receptor having a high affinity for naloxone, previously termed a mu-receptor. The affinity of this receptor for normorphine appears to be 3 to 4 orders or magnitude lower than its affinity for naloxone.

Two affinity states of Ri adenosine receptors in brain membranes. Analysis of guanine nucleotide and temperature effects on radioligand binding.

Abstract: The binding of agonists and antagonists to Ri adenosine receptors of synaptosomal membranes from rat and bovine brain was studied. The effects of guanine nucleotides and temperature were analyzed with the aid of computerized curve fitting. Evidence is presented for two different states of the receptor: one of high and one of low affinity for agonists. Antagonists bind to both states with the same affinity. The two states are characterized by saturation, competition, and kinetic experiments with very similar results. Guanine nucleotides cause transition of the high- to the low-affinity state. The ratio of the KD values for the two affinity states is 90-150 in rat brain but only 10 in bovine brain. The proportions of the two affinity states are the same for all agonists tested; in the absence of exogenous guanine nucleotides, 75% of the total receptor population is in the high-affinity state, whereas in the presence of guanine nucleotides only 5% remain in the high-affinity state. Binding of antagonists to the receptor is enthalpy-driven whereas binding of the agonist (-)-N6-phenylisopropyladenosine to the high-affinity state of the receptor is entropy-driven. Binding of the agonist to the low-affinity state is enthalpy-driven and thus similar to the binding of antagonists. Our data indicate that guanine nucleotides convert the Ri adenosine receptor from a high- to a low-agonist affinity state and that agonist binding shows thermodynamic differences from antagonist binding only when it is to the high-affinity state of the receptor.

Structural gene products of the Ah locus. Transcriptional regulation of cytochrome P1-450 and P3-450 mRNA levels by 3-methylcholanthrene.

Abstract: Mouse liver cytochromes P1-450 and P3-450 represent those forms of polycylic hydrocarbon-induced P-450 most closely associated with induced aryl hydrocarbon (benzo[a] pyrene) hydroxylase and acetanilide 4-hydroxylase activity, respectively. These two proteins are controlled by the Ah receptor: C57BL/6N mice possess the high-affinity receptor; DBA/2N mice, the poor-affinity receptor. 3-Methylcholanthrene at the highest dose technically possible induces both proteins in C57BL/6N but not DBA/2N mice, whereas sufficiently high doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce both proteins in both inbred mouse strains. Plasmids containing DNA complementary to P1-450 and P3-450 mRNA, respectively, were used in an in vitro nuclear transcription assay to determine the mechanism of the induction response. In C57BL/6N mice, transcriptional rates of the P1-450 and P3-450 genes increase dramatically as early as 3 hr after 3-methylcholanthrene treatment and at 12 hr reach maximal levels of 20- and 15-fold, respectively, above control values. In contrast, no increase in either gene is found in 3-methylcholanthrene-treated DBA/2N mice. Following TCDD administration, both P1-450 and P3-450 gene transcription rates are elevated in DBA/2N mice. There is a 3- to 6-hr lag period between the early onset of enhanced transcription rates and the later rise in P1-450 and P3-450 mRNA. Basal and induced levels of P3-450 mRNA are about 5-fold greater than those of P1-450 mRNA. These data confirm that the 3-methylcholanthrene and TCDD induction responses, governed by the Ah receptor, are mediated principally through an increase in specific gene transcription.

Physical properties and lipid composition of brain membranes from ethanol tolerant-dependent mice

Abstract: DBA/2 mice were made tolerant to and dependent on ethanol by administration of an ethanol-containing liquid diet for 7 days. Fluorescent probe molecules were used to estimate the fluidity and ethanol sensitivity of brain synaptic membranes from these mice. The fluorescence polarization of cis- parinarate , trans- parinarate , and 1,6-diphenyl-1,3,5-hexatriene (probes of the membrane core) and 1-(4- trimethylammoniumphenyl )-6-phenyl-1,3,5-hexatriene (a probe of the membrane surface) was higher in membranes from ethanol tolerant-dependent mice than in membranes from control mice. The decrease in fluorescence polarization produced by in vitro exposure to ethanol was attenuated in membranes from ethanol tolerant-dependent mice when 1,6-diphenyl-1,3,5-hexatriene was used as the probe, but not when 1-(4- trimethylammoniumphenyl )-6-phenyl-1,3,5-hexatriene was used. These results indicate that chronic ingestion of ethanol decreased the fluidity and the ethanol sensitivity of the synaptic membranes. In contrast to the alterations observed with intact membranes, liposomes of lipids extracted from synaptic membranes of control and ethanol tolerant-dependent mice did not differ in their physical properties. Analysis of membrane lipids demonstrated that chronic ethanol treatment selectively decreased the unsaturated acyl groups of phosphatidylserine without altering the acyl composition of other phospholipids or sphingolipids. The amount of each phospholipid was not changed, but membrane cholesterol was decreased by chronic ethanol ingestion. Use of 2-dimensional thin-layer chromatography allowed the quantitation of 10 different gangliosides. The concentrations of these lipids were unchanged in synaptic membranes from ethanol tolerant-dependent mice. Thus, the changes in membrane physical properties produced by chronic ingestion of ethanol may be due, at least in part, to altered acyl composition of phosphatidylserine. The differences observed between intact membranes and extracted lipids suggest, however, that chronic ethanol treatment also produced changes in the lipid arrangement or lipid-protein interactions of the intact membranes.

The ternary complex model: its properties and application to ligand interactions with the D2-dopamine receptor of the anterior pituitary gland

Abstract: Agonists and antagonists interact with the pituitary D2-dopamine receptor in a complex fashion that has been accounted for by proposing that the receptor exists in two interconvertible affinity states [De Lean et al., Mol. Pharmacol. 22:290-297 (1982)]. These two states appear to be modulated by guanine nucleotides such that the state existing in the presence of excess guanine nucleotide has low affinity for agonists and high affinity for antagonists. These observations, together with several lines of evidence from other laboratories, have suggested the interaction of the receptor with a guanine nucleotide-binding protein and a model describing the reversible interaction of the receptor (R) with an additional membrane component (X) was studied [De Lean et al., J. Biol. Chem. 255:7108-7117 (1980)]. Several properties of this ternary complex model are presented and discussed in terms of the interpretation of the analysis of simulated binding data using the mass-action model. Computer modeling of experimental binding data obtained from membrane homogenates of bovine anterior pituitary glands indicated that a ternary complex model will fit only under conditions where, in the absence of any ligand, there is a tight interaction or "precoupling" of R with X, with the latter being in stoichiometrically limiting amounts; antagonists and guanine nucleotides would tend to destabilize this interaction, whereas agonists would serve to stabilize the coupled form. These results, for a receptor system that inhibits adenylate cyclase activity, are notably different from those observed for the beta-adrenergic receptor, which stimulates the enzyme and may be a reflection of differences in the molecular mechanisms of the interaction of the two receptor systems with their ligands and their effector. Some features of the model are not compatible with the experimental data and have indicated the need to consider extensions of the model, in light of recent advancements in our understanding of these regulatory components. Our results stress the importance of verifying the properties of proposed models and of cautiously testing these proposed models by their direct application to experimental data.

Comparison of the sodium dependency of uptake of meta-lodobenzylguanidine and norepinephrine into cultured bovine adrenomedullary cells.

Abstract: Radioiodinated meta-iodobenzylguanidine (MIBG), a scintigraphic agent used for the detection of human pheochromocytomas, is thought to utilize the same uptake and retention mechanism(s) as norepinephrine (NE). Using cultured bovine adrenomedullary cells, we compared the mechanism(s) of uptake of MIBG to that of NE. Two different uptake systems were identified. NE and MIBG were taken up by a sodium-dependent system that was characterized by: 1) temperature dependency; 2) high affinity: Km of 1.22 +/- 0.12 microM for MIBG and 1.41 +/- 0.50 microM for NE; 3) low capacity: Vm (picomoles/10(6) cells/10 min) of 64.3 +/- 3.3 for MIBG and 36.6 +/- 7.2 for NE; 4) saturability; 5) ouabain sensitivity; and 6) energy dependency. However, NE and MIBG also were taken up by a temperature-dependent, sodium-independent, apparently unsaturable, and energy-independent system. The sodium-dependent uptake system fulfills many of the criteria for Uptake1 whereas the sodium-independent uptake system is most likely a passive diffusion process. NE uptake proceeded predominantly by the sodium-dependent process. Uptake of MIBG occurred by both pathways at low concentrations, but at high concentrations (greater than 10 microM) uptake was predominantly (75 to 100%) by the sodium-independent process. Inhibition studies suggest that MIBG and NE are transported by the same carrier involved in the sodium-dependent system. Scintiscans of the human adrenals and pheochromocytomas appear to reflect uptake of [131I]MIBG by the sodium-dependent system.

Cytosolic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin. Evidence for a homologous nature among various mammalian species.

Abstract: The presence and properties of the Ah receptor were examined in the guinea pig, rat, hamster, monkey, and three different strains of mice. These species and strains have demonstrated differences in sensitivity and variability of response to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. All species examined, with the exception of DBA/2J mice, possess similar amounts of binding protein with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin in hepatic tissue. Numerous dibenzo-p-dioxin congeners and polycyclic aromatic hydrocarbons demonstrated a similar rank order ability to bind to receptor molecules from these species. When analyzed by gel-exclusion high-performance liquid chromatography, hepatic cytosolic receptors from all species eluted at volumes corresponding to a similar molecular weight range. Association of the hepatic Ah receptor with the nuclear fraction was observed in all cases following the i.p. treatment of guinea pig, rat, C57BL/6J mouse, or hamster with [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin. In all species and tissues examined, with the exception of hamster duodenum and thymus, the highest concentrations of receptor were localized in the liver, lung, thymus, intestine, and kidney. Exceptionally high concentrations of receptors were also observed in guinea pig testes. These findings indicate that, despite species and tissue specific differences in the biochemical and toxicological responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds, a number of different mammalian species possess Ah receptors with similar properties. Thus, the correlative differences between certain strains of mice in terms of altered specific binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin and sensitivity to this compound may be unique and not necessarily applicable to other species. Although all data indicate that the receptor mediates these responses, it appears that species- and tissue-specific differences may be determined by a number of additional factors. These results also suggest the conservation of some, as yet unknown, functional role of the receptor molecule.

Correlation between chemical structure, receptor binding, and biological activity of some novel, highly active, 16 alpha, 17 alpha-acetal-substituted glucocorticoids.

Abstract: The affinity for the glucocorticoid receptor in rat skeletal muscle of some glucocorticoids with a new type of 16 alpha, 17 alpha-acetal substituent has been estimated and correlated to the glucocorticoid activities in three in vivo systems in rats. Budesonide (an approximately 1:1 mixture of the C(22) epimers of 11 beta, 21-dihydroxy-16 alpha, 17 alpha-[(22R,S)-propylmethylenedioxy]-pregna-1,4-diene-3,20-dione) and the isolated (22R)- and (22S)-epimers bound to the same binding site as the potent glucocorticoids dexamethasone (DEX) or triamcinolone 16 alpha, 17 alpha-acetonide (TA), but with even higher affinity than DEX or TA, despite the lack of a 9 alpha-fluoro atom in budesonide and its epimers. The (22R)-epimer was twice as active as the (22S)-epimer, 4 times more active than TA, and 14 times more active than DEX. The introduction of a 9 alpha-fluoro atom slightly decreased the binding affinity of the (22R)-epimer of budesonide, in contrast to the positive effect of 9 alpha-fluorination of, e.g., 16 alpha, 17 alpha-acetonides. The negative influence of 9 alpha-fluorination of the (22R)-epimer was partially reversed in the 6 alpha, 9 alpha-difluorinated (22R)-epimer. Nevertheless, the fluorinated compounds were more active than DEX and TA (8 and 11 times more active than DEX, and 2 and 3 times more active than TA, in case of the 9 alpha-fluoro- and 6 alpha, 9 alpha-difluoro-derivatives of the (22R)-epimer, respectively). Budesonide is metabolized mainly to 16 alpha-hydroxyprednisolone (11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) and 6 beta-hydroxy-budesonide. Both metabolites were very weak competitors for the ligand-binding sites on the receptor (3% and 6% of the affinity of DEX, respectively). The affinity for the receptor in vitro was closely correlated to the topical glucocorticoid activity in vivo for the 12 steroids compared (r = 0.98; R = 0.98), which supports the contention that in vitro tests for receptor affinity are useful when screening for agonists among steroids with the present type of structures. The results on receptor-ligand interaction are in accordance with X-ray crystallographic data available for some steroids.

Inhibition of voltage-sensitive sodium channels in neuroblastoma cells and synaptosomes by the anticonvulsant drugs diphenylhydantoin and carbamazepine.

Abstract: The inhibitory action of a number of clinically effective anticonvulsants on neurotoxin-activated sodium channels in cultured neuroblastoma cells and rat brain synaptosomes has been examined. Diphenylhydantoin (KI = 35 microM) and carbamazepine (KI = 41 microM) inhibited batrachotoxin-activated 22Na+ influx in N18 cells. Similarly, batrachotoxin-activated 22Na+ influx in rat brain synaptosomes was also inhibited by diphenylhydantoin (KI = 38 microM) and carbamazepine (KI = 22 microM). Comparison of KI values with mean brain levels of these drugs achieved during prevention of electroshock seizures indicates that diphenylhydantoin and carbamazepine occupy 35% and 50%, respectively, of their receptor sites associated with sodium channels at mean therapeutic concentrations. Diazepam (KI = 51 to 63 microM) and phenobarbital (KI = 1.2 to 1.3 mM) inhibited batrachotoxin-activated 22Na+ flux in N18 cells and synaptosomes at concentrations in excess of mean therapeutic central nervous system levels. Carbamazepine, like diphenylhydantoin, acts as a competitive inhibitor of sodium channel activation by the full agonist batrachotoxin, but produces mixed inhibition of veratridine-activated channels. This finding is consistent with the conclusion that both carbamazepine and diphenylhydantoin act as allosteric inhibitors of neurotoxin-activated sodium channels. The dose-response relationships for carbamazepine and diphenylhydantoin inhibition of 22Na+ flux in N18 cells are shifted 1.5-fold to higher concentrations when 22Na+ flux measurements are made in the presence of physiological concentrations of sodium and calcium ions. These results suggest that anticonvulsant inhibition of neurotoxin-activated 22Na+ flux in our standard ion flux media, containing low concentrations of Na+ and no Ca2+, is likely to reflect an effect of these agents expected in vivo. The results of this study provide further evidence to support the hypothesis that diphenylhydantoin and carbamazepine, both of which possess similar therapeutic profiles in the treatment of grand mal and partial seizures, may exert their pharmacological effects by occupancy of receptor sites associated with the activation of voltage-sensitive sodium channels in the central nervous system.

Reserpine binding to bovine chromaffin granule membranes. Characterization and comparison with dihydrotetrabenazine binding.

Abstract: [3H]Reserpine bound reversibly in vitro to chromaffin granule membranes. Binding was temperature-dependent and slow, and had biphasic kinetics. The addition of ATP accelerated the kinetics, which became monophasic and comparable to those of [3H] dihydrotetrabenazine, without affecting the binding equilibrium constants. The ATP effect was related to H+ -electrochemical gradient generation by the granule membrane H+ pump. Binding of reserpine to chromaffin granule membranes occurred on two classes of sites: R1, Bmax = 7 pmoles/mg of protein and KD = 0.7 nM, and R2, Bmax = 60 pmoles/mg of protein and KD = 25 nM. Sites R2 were considered to be equivalent to [3H] dihydrotetrabenazine binding sites, as the densities of the R2 and the [3H] dihydrotetrabenazine binding sites were similar and because tetrabenazine displaced reserpine from R2 sites. Sites R1 were tetrabenazine-resistant; they were involved in monoamine uptake, since their KD values were similar to the KI values of reserpine for noradrenaline uptake. Sites R1 were less abundant than sites R2 on chromaffin granule membranes, but they were present at the same concentration in intact chromaffin granules. We propose that the monoamine carrier exists in two forms: (a) an active form bearing both high- and low-affinity sites for reserpine and (b) an inactive form with only the low-affinity R2 sites.

Differential induction of two 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible forms of cytochrome P-450 in extrahepatic versus hepatic tissues.

Abstract: The present study examines the induction of two isozymes of cytochrome P-450, P-448HCB and P-448MC, by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in hepatic and a number of extrahepatic tissues of male rats. These isozymes were quantitated by radioimmunoassay (RIA). TCDD induces both forms of cytochrome P-448 markedly in liver. In extrahepatic tissues, TCDD induces cytochrome P-448MC but not cytochrome P-448HCB. Induction of cytochrome P-448MC is greatest in liver greater than kidney greater than lung greater than intestine greater than spleen greater than testes greater than brain (no significantly increased). The results in liver, kidney, and lung were confirmed by a technique that depends on both electrophoretic mobility and immunological characteristics (sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and immunostaining of the protein). Cytochrome P-448HCB is a minor constitutive form in livers of control male rats (5% of the total cytochrome P-450). In contrast, cytochrome P-448MC is below the level of detection in control livers using the immunostaining technique (less than 0.6% by RIA). These results indicate that induction of cytochrome P-450 isozymes by TCDD is tissue-dependent in the rat. The response of extrahepatic tissues to TCDD is more limited than that of liver.

Relationships between phosphoinositide and calcium responses to muscarinic agonists in astrocytoma cells.

Abstract: Activation of muscarinic receptors in human astrocytoma (1321N1) cells stimulates phosphoinositide metabolism and calcium mobilization. The muscarinic effect on phosphoinositide turnover is evidenced by increased formation of [3H]inositol 1-phosphate (Ins1P) and by increased [3H]inositol incorporation into PtdIns. The muscarinic effect on calcium mobilization is seen as a large increase in undirectional 45Ca2+ efflux from cells equilibrated with 45Ca2+ and a small increase in unidirectional 45Ca2+ influx. A series of muscarinic agonists was used to explore the relationship between phosphoinositide metabolism and unidirectional 45Ca2+ efflux. The maximal increases in [3H]Ins1P formation produced by carbachol and acetylcholine are similar and are much larger than those caused by oxotremorine and pilocarpine. The effects of these agonists on 45Ca2+ efflux are similar: carbachol and acetylcholine cause equivalent maximal increases in the rate of 45Ca2+ efflux whereas oxotremorine and pilocarpine cause submaximal 45Ca2+ efflux responses. The Kact values of carbachol and acetylcholine for stimulation of [3H]Ins1P formation are 40 microM and 1.5 microM, respectively. These values are only 2- to 3-fold higher than the respective Kact values for stimulating 45Ca2+ efflux. The finding that each of the muscarinic agonists tested has nearly identical efficacy and similar potency for stimulating [3H]Ins1P formation and 45Ca2+ efflux supports the idea that hormonal stimulation of phosphoinositide hydrolysis leads to calcium mobilization.

A new type of tachykinin binding site in the rat brain characterized by specific binding of a labeled eledoisin derivative.

Abstract: A new ligand for investigating tachykinin-binding site subtypes was synthesized by coupling the 125I-Bolton and Hunter reagent to eledoisin (125I-BHE). Using a synaptosomal preparation (P2 fraction) of rat cerebral cortex, 125I-BHE was shown to bind with apparent high affinity (apparent Kd = 15.3 nM). When concentrations of up to 30 nM 125I-BHE were used, 125I-BHE binding was specific, saturable, reversible, and temperature-dependent. In contrast to [3H]dopamine, 125I-BHE was not taken up within synaptosomes by an ouabain-sensitive process. Eledoisin, kassinin, and substance P were examined for their ability to inhibit specific 125I-BHE binding to cortical synaptosomes. Eledoisin and kassinin were considerably more potent than substance P, in contrast to the order of potency observed for specific 125I-Bolton-Hunter substance P (125I-BHSP) binding. Specific 125I-BHE binding was highest in the cerebral cortex and hypothalamus; intermediate in the hippocampus, striatum, and thalamus; low in the mesencephalon, septum, and substantia nigra; and absent in the cerebellum. Comparison of these data with those previously obtained for 125I-BHSP binding to synaptosomes indicated that 125I-BHE-labeled binding sites differ markedly from those of 125I-BHSP-labeled binding sites. Therefore, tachykinin receptors other than substance P receptors seem to be present in the central nervous system.

Effects of 1-beta-D-arabinofuranosylcytosine incorporation on eukaryotic DNA template function.

Abstract: 1-beta-D-Arabinofuranosylcytosine (ara-C) incorporates into DNA, and the extent of this incorporation correlates significantly with inhibition of DNA synthesis. The incorporated ara-C residue provides a poor primer terminus for further chain elongation. There is a highly significant relationship between formation of (ara-C) DNA and loss of clonogenic survival. The present studies confirm that incorporation of ara-C into DNA, and not the competitive inhibition of DNA polymerase, is responsible for inducing lethal cellular events. The results also demonstrate that the incorporated ara-C residue is not excised from the DNA strand. Furthermore, the presistence of ara-C residues in DNA inhibits recovery of DNA synthesis following exposure to drug. The relative DNA chain-terminating effect of ara-C provides several mechanisms of action that explain internucleotide and chain terminus positioning of ara-C residues, reinitiation of previously replicated DNA segments, and DNA strand or chromosomal breaks. The precise mechanism of action is dependent upon dose scheduling of this drug.

Albumin binding of anti-inflammatory drugs. Utility of a site-oriented versus a stoichiometric analysis.

Abstract: Binding equilibria of 12 nonsteroidal, anti-inflammatory substances, salicylic acid, diflunisal, phenylbutazone, azapropazone, fenbufen, biphenylacetic acid, naproxen, flurbiprofen, ibuprofin, diclofenac, indomethacin, and benoxaprofen, to defatted human serum albumin has been investigated at 37 degrees, pH 7.4, in a sodium phosphate buffer, 66 mM, by means of equilibrium dialysis and, in case of salicylic acid, by dialysis rate determinations. Cobinding of each of these drugs with monoacetyl-4,4'-diaminodiphenyl sulfone, warfarin, and diazepam has been studied by measuring dialysis rates of the last-mentioned ligands. Cobinding of each drug with bilirubin was investigated by two techniques, equilibrium dialysis against albumin with and without bilirubin, and by measuring rates of oxidation of free bilirubin with hydrogen peroxide and peroxidase. Results were analyzed in quantitative terms. The use of a site-oriented description versus a stoichiometric analysis is discussed. The stoichiometric description is preferred for the following reasons: (a) Simple relations exist between the percentage of bound drug at low drug concentrations and the first stoichiometric binding constant. (b) The stoichiometric description does not imply that preformed binding sites are present in the albumin molecule. (c) A quantitative, stoichiometric analysis of multiple cobinding of two ligands is possible.

Mechanism of the stimulation of prostaglandin H synthase and prostacyclin synthase by the antithrombotic and antimetastatic agent, nafazatrom.

Abstract: Nafazatrom, an antithrombotic and antimetastatic agent containing a pyrazolone functionality, is a reducing substrate for the peroxidase activity of prostaglandin H (PGH) synthase. Nafazatrom inhibits the hydroperoxide-dependent oxidation of phenylbutazone, stimulates the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid, and is oxidized by microsomal or purified enzyme preparations from ram seminal vesicles. Consonant with the effects of other peroxidase-reducing substrates, nafazatrom stimulates the oxygenation of arachidonic acid to prostaglandin endoperoxides by the cyclooxygenase component of PGH synthase. In addition, nafazatrom causes an elevation in the levels of 6-keto-prostaglandin F1 alpha, the non-enzymatic hydrolysis product of prostacyclin (PGI2) biosynthesized from arachidonic acid by ram seminal vesicle microsomes. Elevation of PGI2 biosynthetic capacity by nafazatrom occurs under conditions in which prostaglandin endoperoxide biosynthesis is maximal, suggesting that nafazatrom has a stimulatory effect on the conversion of prostaglandin endoperoxides to PGI2. Nafazatrom has no effect on the ability of ram seminal vesicle microsomes to convert PGH2 to PGI2 but protects microsomal PGI2 synthase from inactivation by 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Nafazatrom stimulates PGI2 biosynthesis in ram seminal vesicle microsomes by acting as a substrate for the peroxidase-catalyzed reduction of hydroperoxy fatty acids that are irreversible inactivators of PGI2 synthase. Several other compounds, including dipyridamole and triiodothyronine, exert similar effects. This may contribute to the reported ability of nafazatrom and related compounds to elevate the levels of bioassayable PGI2 in vivo and to the antithrombotic and antimetastatic activities of nafazatrom.

Biphasic regulation of the gonadotropin-releasing hormone receptor by receptor microaggregation and intracellular Ca2+ levels

Abstract: In the present work we show that Ca2+ is both necessary and sufficient to evoke homologous up-regulation of the gonadotropin-releasing hormone (GnRH) receptor. Extracellular Ca2+ as well as RNA and protein synthesis were required for this event, and it was blocked by Ca2+ ion channel blockers. Drugs which stimulated increased intracellular Ca2+ levels also stimulated receptor up-regulation and enhanced responsiveness even in the absence of added GnRH. Such drugs were effective below the concentrations needed to evoke luteinizing hormone (LH) release, suggesting that enhanced levels of Ca2+ ion, rather than LH depletion, is the responsible agent. A GnRH antagonist did not evoke up- or down-regulation; however, a conjugate of this antagonist, which stimulated microaggregation of the GnRH receptor, also stimulated these biphasic actions. In contrast to up-regulation, down-regulation of the GnRH receptor appears to be Ca2+-independent and does not require RNA or protein synthesis. These data are consistent with a model in which microaggregation of the GnRH receptor is the final step in common to a branched pathway consisting of Ca2+-dependent (LH release, enhanced sensitivity, up-regulation) and Ca2+-independent (desensitization, down-regulation) events.

DT-diaphorase and peroxidase influence the covalent binding of the metabolites of phenol, the major metabolite of benzene.

Abstract: The role of various enzymes and biological molecules on the activation and deactivation of the metabolites of phenol was investigated in vitro. Phenol, the major metabolite of benzene, is metabolized to hydroquinone and catechol. Activation of these metabolites and deactivation of their oxidized forms was assessed by the amount of covalent binding to microsomal protein. [14C]Phenol and NADPH were incubated with hepatic microsomes isolated from phenobarbital-pretreated guinea pigs, and 2.33 nmoles of hydroquinone and 0.12 nmole of catechol were formed per minute per milligram of microsomal protein. Covalent binding of the metabolites to microsomal protein incubated with microsomes isolated from guinea pigs pretreated with phenobarbital was 252 pmoles bound/min/mg; with microsomes from untreated guinea pigs, covalent binding was 146 pmoles bound/min/mg. Covalent binding was inhibited greater than 90% with the addition of N-octylamine, ascorbate, or GSH. The addition of superoxide dismutase inhibited covalent binding with microsomes isolated from phenobarbital-pretreated guinea pigs 35% but did not inhibit it with microsomes isolated from untreated animals. Partially purified guinea pig hepatic DT-diaphorase [NAD(P)H (quinone acceptor) oxidoreductase, EC 1.6.99.2] inhibited covalent binding 70%. This effect was reversed in the presence of dicumarol, a specific inhibitor of DT-diaphorase. DT-diaphorase present in the 10(5) X g supernatant fraction was also active in inhibiting covalent binding but only after the removal of endogenous reduced glutathione. This effect could also be reversed by dicumarol. The addition of diaphorase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) partially purified from Clostridium kluyveri inhibited covalent binding 86%. The addition of hydrogen peroxide and horseradish peroxidase (peroxidase, EC 1.11.17) or myeloperoxidase(s) increased covalent binding 30-fold and 6-fold, respectively. Ascorbate decreased this binding greater than 95%. These results indicate that hydroquinone, catechol, and phenol as well as their oxidized forms can be activated or deactivated by several of the above model systems. These systems may play a role in the myelotoxicity of benzene by modulating covalent binding.

Increased levels of thymidylate synthetase in cells exposed to 5-fluorouracil.

Abstract: The binding of 5-fluoro-2'-deoxyuridylate, methylenetetrahydrofolate, and thymidylate synthetase in a ternary complex results in enzyme inhibition and is a major component of 5-fluorouracil cytotoxicity in some cells. The amount of 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase in several human gastrointestinal tumor cell lines following 5-fluorouracil exposure was determined, using Sephadex G-25 chromatography and high-pressure chromatographic analysis. These data were compared with previously determined values for thymidylate synthetase levels in control cultures not exposed to 5-fluorouracil. In HuTu 80 cells, the amount of 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase represented 16% of the total amount of enzyme present in untreated cells. The values for 5-fluoro-2'-deoxyuridylate bound to thymidylate synthetase in the WIDR and HT 29 cell lines (57 and 46 fmoles/10(5) cells, respectively), however, exceed by 3- to 6-fold the total amount of this enzyme found in untreated cells. The presence of increased levels of thymidylate synthetase protein in these cell lines was confirmed by sodium dodecyl sulfate gel electrophoresis. Measurements of thymidylate synthetase levels following exposure of cells to cycloheximide demonstrated that thymidylate synthetase complexed to 5-fluoro-2'-deoxyuridylate has increased stability as compared with uncomplexed enzyme. The level of thymidylate synthetase (bound and free) present in WIDR cells was measured following removal of FUra from the media. After 48 hr, the level of bound enzyme had fallen from 53 to 14 fmoles/10(5) cells, whereas free enzyme, which was undetectable after a 24-hr exposure to FUra, returned to 60% of its level in untreated cells.

Effects of adenosine dialdehyde on S-adenosylhomocysteine hydrolase and S-adenosylmethionine-dependent transmethylations in mouse L929 cells.

Abstract: Adenosine dialdehyde (2'-O-[(R)-formyl( adenin -9-yl)methyl]-(R) -glyceraldehyde), which is formed by periodate oxidation of adenosine, was shown to be a potent inhibitor of S- adenosylhomocysteine hydrolase (AdoHcy hydrolase; EC 3.3.1.1) in mouse L929 cells. The inhibitory effects of adenosine dialdehyde on AdoHcy hydrolase were time-dependent, having a rapid onset with complete inhibition occurring within a 15-min incubation period. When mouse L929 cells were preincubated with adenosine dialdehyde for 15 min, then transferred to fresh medium, the AdoHcy hydrolase activity returned to control values within 16 hr. When cycloheximide, an inhibitor of protein synthesis, was included in the incubation medium, recovery of AdoHcy hydrolase activity was not prevented, suggesting that the recovery of enzyme activity was probably due to slow reversal of the inhibitor-enzyme complex. The inhibition of AdoHcy hydrolase by adenosine dialdehyde resulted in a time-dependent increase in endogenous AdoHcy levels. After an initial 15-min lag time, the concentration of AdoHcy continued to increase, reaching a maximum of 1200 pmoles/mg of protein in 12 hr. A slight increase in AdoMet levels was observed. Incubation of mouse L929 cells with adenosine dialdehyde also caused an inhibition of lipid methylation and protein carboxylmethylation which resulted from the compound's effect on AdoHcy hydrolase and the subsequent buildup of endogenous AdoHcy levels. Under the conditions that produce inhibition of AdoHcy hydrolase and AdoMet-dependent methyltransferases, adenosine dialdehyde had no effect on RNA or DNA synthesis. Therefore, adenosine dialdehyde appears to be a useful chemical probe with which to study the physiological role of AdoMet-dependent methylations.

Muscarinic cholinergic receptors of two cell lines that regulate cyclic AMP metabolism by different molecular mechanisms.

Abstract: The attenuation of cyclic AMP accumulation occurs by different mechanisms in 1321N1 astrocytoma cells and NG108-15 neuroblastoma X glioma cells. In 1321N1 cells, cholinergic agonists reduce cyclic AMP accumulation through a Ca2+-dependent activation of phosphodiesterase; in NG108-15 cells, muscarinic receptor-mediated effects on cyclic AMP metabolism occur through inhibition of adenylate cyclase. The goal of the current study was to determine whether different pharmacological specificities were expressed by the muscarinic receptor populations of these two cell lines. The affinity of muscarinic receptors for [3H]quinuclidinyl benzilate (6 pM), [3H]N-methylscopolamine (50 pM), and atropine (80 pM) was similar in membrane preparations from each cell line. The affinity of the antagonist, pirenzepine, which has been proposed to be a selective ligand for a muscarinic receptor subtype, was 3-fold higher in competition binding assays carried out with membranes of 1321N1 cells, than with NG108-15 cells. The Hill coefficients of pirenzepine competition curves were not significantly different from unity in both cell lines. This selectivity of pirenzepine was also apparent in studies of the competitive inhibition of carbachol-induced attenuation of cyclic AMP accumulation in intact cells. Differences in the relative affinities of agonists were observed in competition binding analyses carried out with membranes in the presence of GTP and absence of Mg2+. The Ki values of bethanechol and carbachol were 5- and 12-fold lower for receptors of NG108-15 cells than those of 1321N1 cells and the Ki of methacholine was 3.5-fold lower for 1321N1 cells than for NG108-15 cells. The affinities of oxotremorine and arecoline were similar between the two cell lines. These differences in agonist affinities between the two cell lines were much smaller in analyses of muscarinic receptor-mediated effects on cyclic AMP metabolism in intact cells. Taken together, these data suggest that muscarinic receptors of differing pharmacological specificities regulate cyclic AMP metabolism by different mechanisms in 1321N1 and NG108-15 cells.