Sulfation of mono- and diaryl oximes by aryl sulfotransferase isozymes.

Aryl sulfotransferases (3'-phosphoadenylsulfate:phenol sulfotransferase, EC 2.8.2.1) catalyze the sulfonation of a wide variety of hydroxyl-containing substrates, including numerous xenobiotics. The chemical diversity of aryl sulfotransferase substrates is in part attributable to the presence of multiple isozymes, each of which has broad substrate specificity. Of the aryl sulfotransferase isozymes in rat liver cytosol, two (designated isozymes I and II) have previously been shown to sulfonate phenolic compounds exclusively and, moreover, have very similar substrate specificity patterns. The recently reported unusually efficient, rapid isozyme I-catalyzed sulfonation of 9-fluorenone oxime (Mangold, J.B., Mangold, B.L.K. and Spina, A. (1986) Biochim. Biophys. Acta 874, 37-43) was therefore unexpected and suggested that aryl oximes may represent a useful class of model compounds to probe isozymic differences in substrate steric and electronic requirements. In the present study, several mono- and diaryl oximes have been prepared and tested as potential substrates for partially purified aryl sulfotransferases I and II from rat liver cytosol. The results indicate that steric factors, specifically planarity and hydroxyl group position, appear to be important requirements for enzyme-catalyzed sulfonation. In addition, although isozymes I and II had comparable activity with diaryl oximes, some striking differences in the ability of these two isozymes to sulfonate both substituted and unsubstituted monoaryl oximes were observed. This dissimilarity is consistent with distinct differences in the active sites of these isozymes.

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.

Selective protein arylation by acetaminophen and 2,6-dimethylacetaminophen in cultured hepatocytes from phenobarbital-induced and uninduced mice. Relationship to cytotoxicity.

To evaluate the mechanistic importance of covalent binding in acetaminophen (APAP)-induced hepatotoxicity, we compared the effects of 2,6-dimethylacetaminophen (2,6-DMA) to those of APAP in primary cultures of mouse hepatocytes. Immunochemical analysis of electrophoretically separated proteins has shown that the majority of covalent binding after a cytotoxic dose of APAP occurs on two major bands of 44 and 58 kD (Bartolone et al., Biochem Pharmacol 36: 1193-1196, 1987). At equimolar concentrations, 2,6-DMA bound proteins only 15% as extensively as did APAP and was not cytotoxic in hepatocytes from uninduced mice. However, when the hepatocytes were obtained from phenobarbital-induced mice, APAP administration resulted in increased protein arylation and a more rapid onset of cytotoxicity. Furthermore, in the cells from phenobarbital-induced mice, 2,6-DMA not only resulted in increased binding but also in overt cytotoxicity. Since our affinity-purified anti-APAP antibody did not cross-react with 2,6-DMA, a new antibody specific for 2,6-DMA was prepared and, after affinity purification, was used to detect 2,6-DMA protein adducts by Western blotting. Results indicated that, in hepatocytes from both phenobarbital-induced and non-induced mice, the binding of 2,6-DMA was also highly selective with the most prominent target being the 58 kD cytosolic protein. However, by contrast to APAP, only minimal binding to the 44 kD protein was detected after 2,6-DMA treatment. Although several additional protein adducts were increased in treated cells from phenobarbital-induced mice, the 58 kD protein was clearly the most prominently arylated target associated with both APAP and 2,6-DMA cytotoxicity. These data suggest that both the specificity of covalent binding as well as the extent of binding to the major targets may play an important role in the ensuing toxicity.

Haloperidol competitively inhibits the binding of (+)-[3H]SKF-10,047 to sigma sites.

Scatchard plots of equilibrium saturation binding data revealed that haloperidol inhibits the binding of (+)-[3H]SKF-10,047 to sigma sites in a competitive manner. In experiments using membranes from both guinea pig and rat brain the apparent Kd of (+)-[3H]SKF-10,047 for sigma sites was significantly increased, whereas the apparent Bmax was not altered by the addition of 10 nM haloperidol.

Haloperidol-sensitive (+)[3H]SKF-10,047 binding sites (sigma sites) exhibit a unique distribution in rat brain subcellular fractions.

The distribution of haloperidol-sensitive (+)[3H]N-allylnormetazocine ((+)[3H]SKF-10,047) binding sites (sigma sites) in subcellular fractions of rat brain homogenates was extensively characterized. In synaptosomal fractions, enriched in choline acetyltransferase activity, sigma sites were present in lower concentrations than in whole brain homogenates. On the other hand, microsomal and myelin fractions were found to be enriched in sigma sites. A similar pattern of enrichment was seen for 5'-nucleotidase activity, a general plasma membrane marker. However, subsequent experiments in which microsomes were subfractionated on linear sucrose gradients led to the recovery of sigma sites over a significantly lower density range than 5'-nucleotidase activity or ATP-stimulated [3H]ouabain binding, an additional plasma membrane marker. In addition, previously reported distributions of a number of other subcellular markers, including those for endoplasmic reticulum, were found to contrast with the observed distribution of sigma sites. It is concluded that rat brain sigma sites are not concentrated at synaptic regions of plasma membrane. However, the possibility that sigma sites are localized to specialized areas of nonsynaptic plasma membrane cannot be excluded.

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.

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.

Use of the radial maze in studies of phencyclidine and other drugs of abuse.

Effects of drugs known to disrupt performance in an 8-arm radial maze are reported in terms of changes caused in the pattern of arm entry. Phencyclidine (PCP) and N-allyl-N-normetazocine (SKF-10,047) alter the pattern of arm entry in a way which distinguishes their actions from those of scopolamine and certain serotonergic agonists. The apparent rank order of potencies for causing this effect is (+)SKF-10,047 greater than PCP greater than (-)SKF-10,047. Results of previous radial maze studies evaluating the interactions of clonidine and verapamil with PCP are summarized. Data are reported which indicate that the ability of verapamil to potentiate PCP's behavioral effects stems from an alteration of the pharmacokinetics of PCP; when verapamil (20 mg/kg, IP) was administered 15 minutes before [3H]PCP (40 microCi/kg, IP), brain levels of tritium were increased by 154 to 225 percent. Finally, possible advantages of using a 4-arm radial maze in studies of PCP and related drugs are discussed.

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.

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.

Effects of phencyclidine, N-allyl-N-normetazocine (SKF-10,047), and verapamil on performance in a radial maze.

Two groups of eight rats were trained to obtain food pellets in an 8-arm radial maze. Stable performance was assumed to be present when a criterion of 89% efficiency, i.e., all arms entered within 9 arm entries, was reached in 5 consecutive sessions. The effects of phencyclidine (PCP) and N-allyl-N-normetazocine (SKF-10,047) were then evaluated in Group 1. The interaction between verapamil and PCP was examined in Group II. Both PCP (6 mg/kg, IP, 15 min before testing) and SKF-10,047 (30 mg/kg, IP, 30 min) decreased efficiency but only PCP caused a concurrent increase in rate of arm entry. Significant effects of PCP on rate and efficiency lasted for greater than 6 hours and less than 40 minutes, respectively. Verapamil (20 mg/kg, IP, 30 minutes) was found to selectively potentiate the effect of PCP on efficiency. This finding does not support the suggestion that verapamil may be useful in the treatment of PCP intoxication. It is concluded that the radial maze may provide an interesting method for the study of PCP and other psychoactive drugs.

Interactions of clonidine with phencyclidine and ketamine: studies of radial maze performance and righting reflex in rats.

Rats were trained to obtain food pellets in an 8-arm radial maze until a criterion of 89% efficiency, i.e., all arms entered within 9 arm entries, was reached in 5 consecutive sessions. Decreases in efficiency caused by phencyclidine (PCP; 4 to 9 mg/kg, IP, 15 min before testing) or ketamine (25 mg/kg, IP, 5 min) were attenuated when subjects were pretreated with clonidine (0.05 mg/kg, IP, 30 min). However, significant improvements in performance in the maze were not observed when clonidine (0.05 to 0.4 mg/kg, IP) was administered 15 min after PCP (9 mg/kg, IP, 45 min). Subsequent studies of righting reflex demonstrated an increased frequency and duration of anesthesia when clonidine (0.05 mg/kg, IP) was administered 15 minutes before PCP (12.5 to 50 mg/, IP) or ketamine (50 to 100 mg/kg, IP). When clonidine (0.05 mg/kg, IP) was administered 15 minutes before [3H]PCP (40 microCi/kg, IP), brain levels of tritium were reduced by 42 to 55%. The present findings do not support the suggestion that clonidine may be useful in the treatment of PCP intoxication. The data does indicate that pretreatment of surgical patients with clonidine may reduce the dose of ketamine required for anesthesia.

Phencyclidine/SKF-10,047 binding sites: evaluation of function.

Results of correlation analyses comparing rank-order affinities with rank-order potencies of (+)SKF-10,047, phencyclidine (PCP), and several PCP analogs support the involvement of [3H]-1-[1-(2-thienyl)cyclohexyl]piperidine binding sites (TCP sites) in mediating both the discriminative stimulus properties of PCP and production of 180 degrees perseveration in a 4-arm radial maze. For the same group of drugs, no significant relationship was found to exist between affinities at haloperidol-sensitive (+)[3H]SKF-10,047 binding sites (H-S-SKF sites) and potencies. Also, H-S-SKF sites were found to lack pharmacological selectivity and to be localized in the microsomal fraction of cells. It is concluded that TCP sites may represent receptors which mediate effects not only of PCP, but also of (+)SKF-10,047. In addition, the possibility that H-S-SKF sites may represent a type of membrane-bound enzyme is discussed.

Bilateral arthrography of the wrist.

In order to determine the true significance of a positive arthrogram in the investigation of wrist pain, a prospective trial was carried out in 60 patients, using the opposite asymptomatic wrist as a control. Of the 46 patients with positive findings in the symptomatic wrist, 34 (74%), had positive arthrograms in the opposite, asymptomatic, wrist. As a result of this, we conclude that a unilateral arthrogram is of little diagnostic value and we recommend the use of the opposite, asymptomatic, wrist as a control.

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.

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.

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.

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.