The selective oestrogen receptor modulator, LY362321, is not neuroprotective in a rat model of transient focal ischaemia.

Selective oestrogen receptor modulators (SERMs) may offer improved alternatives to oestrogen as neuroprotectants in experimental stroke. The present study investigated the role of a novel SERM, LY362321, in a rat model of transient middle cerebral artery occlusion (MCAO). Female Sprague-Dawley rats were ovariectomised and began receiving daily s.c. injections of either 1 mg/kg (n = 13), 10 mg/kg (n = 14) of LY362321, or vehicle (n = 13). The left MCA was temporarily occluded (90 min), with cortical blood flow monitoring, at 12 days post ovariectomy. Sensorimotor function was assessed using a neurological score prior to the MCAO and daily for 3 days following the MCAO. Tissue was processed for infarct volume assessment using 2,3,5-triphenyltetra-zolium chloride staining. The results indicated that there were no significant differences amongst groups in cortical blood flow during the MCAO. Furthermore, there was no significant difference in infarct size amongst vehicle, 1, and 10 mg/kg treated animals: 22.9 +/- 5.0, 16.7 +/- 4.2, and 21.1 +/- 4.1, respectively, one-way anova [F(2,32) = 0.542, P = 0.587]. The MCAO induced a significant decline in neurological score in the vehicle group (from 14 to 7 at 24 h post-MCAO) but this was not significantly affected by LY362321 at either dose. In conclusion, pretreatment with a low or high dose of the novel SERM LY362321 did not significantly influence cerebral blood flow, infarct volume, or sensorimotor function in rats exposed to transient MCAO.

Potential of buprenorphine/naltrexone in treating polydrug addiction and co-occurring psychiatric disorders.

In recent years, we have seen regulatory approval being given for several new pharmacotherapies in the treatment of drug addiction disorders. Within the United States, the most noteworthy development has been the approval of buprenorphine in the treatment of opioid dependence, and its availability for prescribing in an office-based setting has resulted in thousands of additional patients going into treatment. Although approved medications for the treatment of cocaine and methamphetamine dependence are still lacking, the National Institute on Drug Abuse has devoted substantial effort toward meeting these clinical needs. Recent studies of modafinil for the treatment of cocaine dependence have been especially encouraging. Looking to the future, the looming challenge is polydrug addiction, a situation that is often complicated by co-occurring psychiatric disorders. As we strive to address the needs of these complicated patients, studies of buprenorphine/naltrexone may hold the key to a major advance.

Body size and V.O2peak: a new perspective?

The purpose of this paper was to show how dimensional analysis and biological similarity theory can be used to define and derive extensive (size-dependent) and intensive (size-independent) variables. The method was then used to analyze the peak rate of oxygen consumption (V.O2peak) data of children, adolescents, and adults to determine if size-independent increases in V.O2peak occur during growth and development. The results indicated growth and development is accompanied by a substantial increase in size-independent V.O2peak, which was associated with improvements in size-independent peak heart rate ( HR(peak)). When differences in fat free mass (FFM) were considered, size-independent HR(peak) was similar in boys and girls within each age-group, but was greater in men than in women. In contrast, when differences in FFM were accounted for, size-independent peak oxygen pulse (O2P(peak)) was independent of age, although males tended to have greater values than females at all ages. Because O2P(peak) is proportional to the product of stroke volume ( SV) and arterial mixed-venous oxygen difference (a-vO2peak), when differences in FFM were considered, the size-independent nature of O2P(peak) indicated the combined effects changes in SV(peak) and (a-vO(2)peak) played a secondary role in improving aerobic capacity. In conclusion, during normal growth and development improvement in HR(peak), not stroke volume and arterial mixed-venous oxygen difference, appears to increase aerobic capacity.

Quaternary ammonium-linked glucuronidation of 1-substituted imidazoles: studies of human UDP-glucuronosyltransferases involved and substrate specificities.

A series of eight 1-substituted imidazoles was investigated as model substrates for glucuronidation at an aromatic tertiary amine of polyaza heterocyclic ring systems. The human UDP-glucuronosyltransferases (UGTs) involved and substrate specificities were investigated. Nine expressed enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) were examined, but only UGT1A4 catalyzed the formation of a quaternary ammonium-linked glucuronide metabolite for six of the substrates. UGT1A3 also catalyzed the glucuronidation of the previously investigated 1-phenylimidazole but none of the newly investigated compounds. No glucuronidation was observed with 1-(4-nitrophenyl)imidazole, the compound with the 4-phenyl substituent with the largest electron withdrawing effect. The incubation conditions for the determination of the kinetic constants for UGT1A4 catalysis of six substrates were optimized and included incubation at pH 7.4 with alamethicin at 10 microg/mg of protein. Latency disrupting agents, including alamethicin and sonication, enhanced glucuronidation 1.25-fold at most. There were 17.5- and 2.2-fold variations in the apparent K(m) (range, 0.18-3.15 mM) and V(max) values (range, 0.16-0.35 nmol/min/mg of protein). Linear correlation analyses between UGT1A4 kinetics and substrate physicochemical parameters showed significant correlation between V(max) and both the partition coefficient (log P, n-octanol/water) and pK(a) and between K(m) and pK(a), thereby indicating that the lipophilicity and the ease of availability of the tertiary amine lone pair of electrons of the substrate are important with respect to enzyme catalysis.

Synthesis and identification of the quaternary ammonium-linked glucuronide of 1-phenylimidazole in human liver microsomes and investigation of the human UDP-glucuronosyltransferases involved.

1-Phenylimidazole was investigated as a potential model substrate with respect to formation of a quaternary ammonium-linked glucuronide (N(+)-glucuronide) at an aromatic type tertiary amine. A reference sample of the potential N(+)-glucuronide metabolite of 1-phenylimidazole was obtained by organic synthesis. The structural identity of the metabolite formed by incubation of 1-phenylimidazole with human liver microsomes was proven to be the N(+)-glucuronide by exhibiting the same HPLC retention time and electrospray ionization mass spectrum as the reference sample. The screening of 1-phenylimidazole against a panel of nine expressed human UDP-glucuronosyltransferases indicated the involvement of UGT1A3 and UGT1A4 in the formation of the N(+)-glucuronide metabolite.

Effects of clofibrate and indocyanine green on the hepatobiliary disposition of acetaminophen and its metabolites in male CD-1 mice.

1. The effects of clofibrate (CFB) and indocyanine green (ICG) on the biliary excretion of acetaminophen (APAP) and its metabolites were investigated. 2. Male CD-1 mice were pretreated with 500 mg CFB/kg, i.p. for 10 days. Controls received corn oil vehicle only. After overnight fasting, common bile duct-cannulated mice were challenged with a non-toxic dose of APAP (1 mmol/kg, i.v.). 3. CFB pretreatment did not affect bile flow rate, nor did it affect the cumulative biliary excretion of APAP and its conjugated metabolites. 4. Additional CFB or corn oil pretreated mice were given 30 mumol indocyanine green (ICG)/kg, i.v., immediately before APAP dosing. ICG is a non-metabolizable organic anion that is completely excreted into the bile through a canalicular transport process for organic anions. 5. ICG significantly decreased the bile flow rate and biliary concentration of APAP-glutathione, APAP-glucuronide and APAP-mercapturate within the first hour after dosing without affecting the biliary concentration of APAP. 6. The results indicate that CFB pretreatment does not affect the total amount of APAP and its metabolites excreted in bile. They also suggest that the biliary excretion of several conjugated metabolites of APAP share the same excretory pathway with the organic anion ICG.

Wet bulb globe temperature index and performance in competitive distance runners.

In 1974 two sets of heat stress guidelines, each based on the wet bulb globe temperature (WBGT) index, were designed for men's National Collegiate Athletic Association (NCAA) Championship Division I distance running competitions. One set of guidelines was established to minimize the chance of heat injury during distance running events. A second set was designed to predict heat stress related performance decrements. During the time the heat injury guidelines were used (1974-1993), no heat injuries were reported. The purpose of this study was to assess the accuracy of the performance decrement guidelines and determine whether the WBGT indices were linearly related to men's championship distance running performance. WBGT index data were collected during the 1500-, 3000-steeplechase (SC), 5000-, and 10,000-m events at men's NCAA Division I Track and Field Championships held from 1974 to 1981 (excluding 1975). These data were compared to the average running performance of the top six finishers in each event. Analysis of the accuracy of the NCAA performance decrement guidelines revealed four unexpected performances out of 28 predictions. Pearson product-moment correlation and linear regression analyses between the WBGT indices and performance revealed statistically significant linear relationships for the 3000-SC and 10,000-m events (P < 0.05). A significant linear relationship was also found when the 1500-, 3000-SC, 5000-, and 10,000-m results were pooled (P < 0.05). In conclusion, the NCAA guidelines were effective in preventing heat injury and fairly successful in predicting performance. However, a linear relationship between WBGT indices and distance running performance did not exist in all running events.

Induction of a permeability transition in rat kidney mitochondria by pentachlorobutadienyl cysteine: a beta-lyase-independent process.

A Ca2+-dependent inner mitochondrial membrane permeability transition is induced by a number of agents, an effect which is thought to cause cytotoxicity. This transition involves formation of a pore allowing the passage of solutes of up to 1500 Da; it is blocked by cyclosporine A and Ca2+ chelating agents. The mitochondrial nephrotoxicant S-(1,2,3,4, 4-pentachlorobutadienyl)-L-cysteine (PCBC) caused collapse of the mitochondrial membrane potential, Ca2+-independent oxidation of pyridine nucleotides and release of accumulated Ca2+ in isolated rat kidney mitochondria, three hallmarks of the permeability transition. These effects were blocked by cyclosporine A and by ethylene glycol bis(beta-aminoethyl ether) tetraacetic acid (EGTA). Furthermore, EGTA was capable of reversing the collapse of the membrane potential. These data indicate that PCBC induced an inner membrane permeability transition. Interestingly, addition of aminoxyacetic acid, a beta-lyase inhibitor, did not prevent the permeability transition, and a nonmetabolizable analog of PCBC, S-(1,2,3,4, 4-pentachlorobutadienyl)-L-alpha-methyl cysteine, induced the permeability transition. Thus PCBC may act to induce the permeability transition through a mechanism that does not require metabolism by a beta-lyase. Since metabolism by a beta-lyase is required for PCBC toxicity, it is not clear that the permeability transition is involved in cysteine conjugate-mediated renal cell injury.

Phosphocreatine kinetics in humans during exercise and recovery.

System linearity was assessed for exercise induced changes in energetics of forearm exercise. 31P-NMR spectroscopy techniques, with 12.5-s serial measurements of [PCr], [Pi], [ATP], and [H+] were employed during exercise and recovery transitions in four untrained men for moderate (1.7 W) and heavy (3.6 W) exercise. Signal averaging was applied and data were analyzed by regression analysis using a first-order exponential model. The time constants for both [PCr] and [Pi] responses to moderate exercise and recovery were not different both within and between nuclei ranging from 32 to 35 s (P > 0.05). The time constants derived from moderate exercise and recovery, when employed to construct predictive equations for heavy exercise and recovery, did not adequately describe [PCr] dynamics. Underestimation of the net hydrolysis of PCr during heavy exercise was associated with increases in [H+] as predicted by the creatine kinase equilibrium reaction (CKeq). Calculation of [ADP] by CKeq revealed steady state [ADP] was achieved during moderate exercise and during recovery for both intensities much earlier than during heavy exercise. We conclude that the metabolic system does not behave as a linear system. Therefore, the time constant and the net change in [PCr].W-1 must themselves be determined by work dependent combinations of other system variables.

Peak torque per unit cross-sectional area differs between strength-trained and untrained young adults.

It is unclear whether gender differences in the relative strength of the upper and lower body are due to differences in muscle mass distribution or dissimilarity of use. There is also controversy as to whether prolonged resistance training increases strength per unit cross-sectional area (CSA). To help resolve these questions, maximum isometric torque per unit muscle and bone (M+B) CSA was measured in the upper arm and thigh of 26 trained (13 males; 13 females) and 26 untrained (13 males; 13 females) young adults. Muscle and bone CSA values were calculated from limb circumferences and skinfolds. Maximal isometric torque values were recorded by a LIDO isokinetic dynamometer. There was no significant difference (P > 0.05) in mean upper arm or thigh torque per unit M+B CSA between the trained males and trained females, or between the untrained males and untrained females. However, mean torque per unit M+B CSA was significantly higher for the trained subjects of both genders compared with the untrained subjects of both genders for the upper arm (28.9%; P < 0.0001) and thigh (18.8%; P < 0.0001). These results suggest that muscle quality (peak torque/CSA) is equal between genders, and that the increase in muscle strength per unit area that occurs with resistance training is not gender-dependent.

Sigma-1 and sigma-2 sites in rat brain: comparison of regional, ontogenetic, and subcellular patterns.

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM(+)[3H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [3H]DTG in the presence of 1 microM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations of sigma-1 and sigma-2 sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely.

Sulfation of di- and tricyclic phenols by rat liver aryl sulfotransferase isozymes.

Aryl sulfotransferases (ASTs) catalyze the sulfation of a variety of hydroxyl-containing substrates, including phenols, aryl oximes, benzylic alcohols, and arylhydroxamic acids. Sulfation of the latter class of substrates (e.g., N-hydroxy-2-acetamidofluorene) can yield highly unstable sulfuric acid esters capable of covalently binding to cellular nucleophiles. Accordingly, these enzymes have been implicated in the bioactivation of the arylhydroxamic acid (and precursor arylamine) class of hepatocarcinogens. Rat liver contains three well-characterized isoforms of AST. To understand better the factors which influence isozymic substrate specificity, the present study focused on steric and regiochemical factors with the sulfation of polyaromatic phenols as a model system. Seven di- and tricyclic phenols were tested as substrates for ASTs I, II, and IV. Based on a comparison of kinetic constants and assuming an absence of substrate-specific pH effects, the results suggest that regiochemical and steric factors play an important role in substrate specificity and provide insight into isozymic differences in active-site topology. For both AST I and AST II, the kinetic results were consistent with an active-site model in which the hydrophobic substrate binding pocket is wider, but less elongated, than that for AST IV. In addition, kinetic results for AST II with 4-phenylphenol were indicative of negative cooperativity which was unique to this isozyme. In contrast to those for ASTs I and II, the kinetic results for AST IV suggest an active-site model that is linearly extended. This elongated active-site model accommodates lengthy substrates and appears to derive little catalytic benefit from additional aromatic rings which increase substrate width.

An absence of changes in sigma receptor subtypes in the brains of genetically dystonic (dt) rats.

Binding sites for the sigma ligand [3H]di-o-tollylguanidine ([3H]DTG) have been reported to be altered in the brains of genetically dystonic rats. In the present study, selective sigma 1 and sigma 2 assay conditions were utilized in an effort to define which subpopulation of [3H]DTG binding sites is altered in the dystonic strain (dt). Both this approach and a re-examination using conditions similar to the previous report failed to confirm a difference between the brains of dystonic and normal rats in terms of sigma binding. Although not directly negating the possible involvement of sigma receptors in dystonia, these results indicate that sigma 1 and sigma 2 receptors appear unchanged in dystonic rats.

Aryl sulfotransferase-IV-catalyzed sulfation of aryl oximes: steric and substituent effects.

The aryl sulfotransferases (EC 2.8.2.1) catalyze the sulfation of a wide variety of hydroxyl-containing molecules. The enzyme reaction requires 3'-phosphoadenosine-5'-phosphosulfate as the sulfate donor and several isozymes with broad, overlapping substrate specificities have been identified. One of the isozymes in rat hepatic cytosol, isozyme IV, is a major contributor to enzymatic sulfation. It exhibits the broadest substrate specificity of the three isozymes which have been characterized to date. Its substrates include a wide variety of phenols, certain aromatic hydroxylamines and benzylic alcohols. The latter two substrate types have implicated this isozyme in the bioactivation of several toxic compounds. Relatively little information is available, however, on substrate molecular features which account for the ability of isozyme IV to sulfate compounds not utilized by isozymes I and II. A recent investigation of isozymes I and II with a series of model aryl-oxime substrates suggested that catalysis is influenced primarily by steric factors and in particular substrate planarity and hydroxyl group orientation (Mangold et al. (1989) Biochim. Biophys. Acta 991, 453-458). In the present study, isozyme IV was investigated to characterize its substrate requirements with a more extensive series of aryl oxime substrates. The results indicated that isozyme IV has a much less stringent requirement for planarity and hydroxyl-group orientation than isozymes I or II. Isozyme IV accepted a greater variety of aryl-oxime substrates, including several classes which were not substrates for isozymes I and II. A comparison of kinetic constants and catalytic efficiencies suggested that substituent effects play a role in the sulfation of aryl oximes by isozyme IV.

Tris inhibits (+)-[3H]SKF-10,047 binding to sigma receptors.

Tris-HCl is the most commonly used buffer in studies of radioligand binding to sigma receptors, with concentrations as high as 50 or 100 mM often used. We report here that these concentrations of Tris substantially inhibit (+)-[3H]SKF-10,047 binding to sigma receptors. The well-established inhibitory effect of Tris-HCl on ligand binding to PCP receptors did not contribute to the presently reported inhibition of (+)-[3H]SKF-10,047 binding. The IC50 of Tris, determined in the presence of 10 mM potassium phosphate buffer, was 15.4 +/- 1.2 mM (n = 3, pH 8.0, 25 degrees C, 1 nM radioligand). Equilibrium saturation studies revealed an apparent competitive inhibition of binding.

Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts.

Antibodies raised against halothane metabolite adducts cross-react with S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine metabolite adducts. Using these antibodies in immunohistochemical experiments, metabolite binding was localized to the damaged areas of the proximal tubule after treatment of male rats with TFEC. Immunoblot analysis of subcellular fractions of rat kidney tissue after in vivo treatment with TFEC revealed a high specificity for binding of metabolites to proteins of the mitochondrial fraction. These proteins may represent target molecules which play a role in cysteine conjugate induced nephrotoxicity.

Formation of difluorothionoacetyl-protein adducts by S-(1,1,2,2-tetrafluoroethyl)-L-cysteine metabolites: nucleophilic catalysis of stable lysyl adduct formation by histidine and tyrosine.

19F NMR spectroscopy was used in conjunction with isotopic labeling to demonstrate that difluorothionoacetyl-protein adducts are formed by metabolites of the nephrotoxic cysteine conjugate S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC). To determine which amino acid residues can be involved in adduct formation, the reactivity of TFEC metabolites with a variety of N-acetyl amino acids was also investigated. An N alpha-acetyl-N epsilon-(difluorothionoacetyl)lysine (DFTAL) adduct was isolated and characterized by 19F and 13C NMR spectroscopy and mass spectrometry. N alpha-Acetylhistidine and N-acetyltyrosine were found to act as nucleophilic catalysts to facilitate the formation of both the protein and DFTAL adducts. Adduct formation was greatly reduced when lysyl-modified protein was used as the substrate, indicating that lysyl residues are primary sites of adduct formation. However N alpha-acetyllysine, at concentrations of greater than 100-fold in excess compared to protein lysyl residues, was not effective in preventing binding of metabolites to protein. Therefore, nucleophilic catalysis at the surface of the protein may be an important mechanism for the binding of TFEC metabolites to specific lysyl residues in protein. TFEC metabolites were very reactive with the thiol nucleophiles glutathione and N-acetylcysteine. However, the predicted difluorodithioesters could not be isolated. Both stable difluorothioacetamide and less stable difluorodithioester protein adducts may play a role in TFEC-mediated nephrotoxicity.

Interactions of [3H]amphetamine with rat brain synaptosomes. I. Saturable sequestration.

Previous studies have identified a saturable site of d-[3H]amphetamine sequestration (AMSEQ) in rat brain synaptosomes. The present study characterized AMSEQ with respect to its subcellular, neuronal and regional distributions, ontogenetic development, pharmacological specificity and factors required for its maintenance. Although AMSEQ was reduced when assays were performed in Krebs' buffer incubated at 37 degrees C as compared to assays performed in isotonic Tris-sucrose buffer incubated at room temperature, the pharmacological profiles of AMSEQ were virtually identical under both conditions. AMSEQ was negligible in tissues outside the central nervous system, enriched in synaptosomes and partially reduced by striatal kainic acid lesion, indicating neuronal localization. The distribution of AMSEQ in the central nervous system was heterogenous. Highest levels were present in hypothalamus with progressively lower levels noted in parietal cortex, frontal cortex, striatum, thalamus, hippocampus, midbrain, cerebellum, pons-medulla and spinal cord. With regard to its ontogeny, AMSEQ increased early in neonatal life, reaching adult levels by postnatal day 14. Although the effects of amphetamine to abolish the transynaptosomal pH gradient suggest a possible role for this gradient in the maintenance of AMSEQ, the pharmacological profile of AMSEQ indicates that other factors are involved. An interaction with an intrasynaptosomal acid, such as N-acetylaspartate, may account for AMSEQ maintenance. AMSEQ did not possess a stereospecific preference for either d-(IC50 = 177 microM) or I-amphetamine (IC50 = 173 microM). However, the pharmacological profile of AMSEQ indicated structural specificity with antidepressants being relatively potent inhibitors. The saturation analysis and pharmacological characteristics of AMSEQ (i.e., low affinity and lack of stereospecificity) suggest that AMSEQ may be related to high dose, nonstereospecific effects of amphetamine, such as stereotypy in rats and psychosis in humans. The relative high potency of antidepressants in inhibiting AMSEQ suggests the importance of this phenomenon in the therapeutic effects of these drugs. Furthermore, the link between antidepressants and AMSEQ may provide insight into the comorbidity of major depression and drug abuse.

Solubilization and characterization of haloperidol-sensitive (+)-[3H]SKF-10,047 binding sites (sigma sites) from rat liver membranes.

The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-[3H]SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-[3H]SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-[3H]SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition, the binding of 20 nM [3H]progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-[3H]SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-[3H]SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex.

Self-catalyzed irreversible inactivation of rat hepatic aryl sulfotransferase IV by N-hydroxy-2-acetylaminofluorene.

Rat hepatic aryl sulfotransferase IV catalyzes the sulfonation of the hepatocarcinogen, N-hydroxy-2-acetylaminofluorene. The resulting reactive N-O-sulfate ester is believed to be the ultimate carcinogenic species responsible for the induction of hepatic neoplasia. Previous studies have shown that dietary administration of either 2-acetylaminofluorene or N-hydroxy-2-acetylaminofluorene to rats is accompanied by a rapid decline in hepatic aryl sulfotransferase activity in vivo. In the present study, preincubation of purified rat hepatic aryl sulfotransferase IV with N-hydroxy-2-acetylaminofluorene resulted in rapid, time-dependent enzyme inactivation. This in vitro inactivation was not reversed by dialysis or gel filtration. Inclusion of excess nucleophile, methionine, resulted in considerable but not complete protection from inactivation. The inactivation was PAPS dependent and blocked by the sulfotransferase inhibitor, pentachlorophenol. The above observations and the apparent pseudo first-order kinetics observed suggest that the inactivation was in part mechanism based. Mechanism-based inactivation of the aryl sulfotransferases has not been previously reported. Furthermore, the results of the present study indicate that the previously reported in vivo decline in rat hepatic aryl sulfotransferase activity may be attributable in part to enzyme inactivation by its own reactive product.