Evaporative isotope enrichment as a constraint on reach water balance along a dryland river.

Deuterium and oxygen-18 enrichment in river water during its transit across dryland region is found to occur systematically along evaporation lines with slopes of close to 4 in (2)H-(18)O space, largely consistent with trends predicted by the Craig-Gordon model for an open-water dominated evaporating system. This, in combination with reach balance assessments and derived runoff ratios, strongly suggests that the enrichment signal and its variability in the Barwon-Darling river, Southeastern Australia is acquired during the process of evaporation from the river channel itself, as enhanced by the presence of abundant weirs, dams and other storages, rather than reflecting inherited enrichment signals from soil water evaporation in the watershed. Using a steady-state isotope mass balance analysis based on monthly (18)O and (2)H, we use the isotopic evolution of river water to re-construct a perspective of net exchange between the river and its contributing area along eight reaches of the river during a drought period from July 2002 to December 2003, including the duration of a minor flow event. The resulting scenario, which uses a combination of climatological averages and available real-time meteorological data, should be viewed as a preliminary test of the application rather than as a definitive inventory of reach water balance. As expected for a flood-driven dryland system, considerable temporal variability in exchange is predicted. While requiring additional real-time isotopic data for operational use, the method demonstrates potential as a non-invasive tool for detecting and quantifying water diversions, one that can be easily incorporated within existing water quality monitoring activities.

The novel, orally active, delta opioid RWJ-394674 is biotransformed to the potent mu opioid RWJ-413216.

Although the mu opioid receptor is the primary target of marketed opioid analgesics, several studies suggest the advantageous effect of combinations of mu and delta opioids. The novel compound RWJ-394674 [N,N-diethyl-4-[(8-phenethyl-8-azabicyclo]3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide]; bound with high affinity to the delta opioid receptor (0.2 nM) and with weaker affinity to the mu opioid receptor (72 nM). 5'-O-(3-[(35)S]-thio)triphosphate binding assay demonstrated its delta agonist function. Surprisingly given this pharmacologic profile, RWJ-394674 exhibited potent oral antinociception (ED(50) = 10.5 micromol/kg or 5 mg/kg) in the mouse hot-plate (48 degrees C) test and produced a moderate Straub tail. Antagonist studies in the more stringent 55 degrees C hot-plate test demonstrated the antinociception produced by RWJ-394674 to be sensitive to the nonselective opioid antagonist naloxone as well as to the delta- and mu-selective antagonists, naltrindole and beta-funaltrexamine, respectively. In vitro studies demonstrated that RWJ-394674 was metabolized by hepatic microsomes to its N-desethyl analog, RWJ-413216 [N-ethyl-4-[(8-phenethyl-8-azabicyclo[3.2.1]oct-3-ylidene)-phenylmethyl]-benzamide], which, in contrast to RWJ-394674, had a high affinity for the mu rather than the delta opioid receptor and was an agonist at both. Pharmacokinetic studies in the rat revealed that oral administration of RWJ-394674 rapidly gave rise to detectable plasma levels of RWJ-413216, which reached levels equivalent to those of RWJ-394674 by 1 h. RWJ-413216 itself demonstrated a potent oral antinociceptive effect. Thus, RWJ-394674 is a delta opioid receptor agonist that appears to augment its antinociceptive effect through biotransformation to a novel mu opioid receptor-selective agonist.

A quantitative genomic expression analysis platform for multiplexed in vitro prediction of drug action.

Genomic expression signatures provide high-content biomarkers of cellular physiology, including the diverse responses to therapeutic drugs. To recognize these signatures, we devised a method of biomarker evaluation called 'sampling over gene space' (SOGS) that imparts superior predictive performance to existing supervised classification algorithms. Applied to microarray data from drug-treated human cortical neuron 1A cell cultures, this method predicts whether individual compounds possess anticonvulsant, antihypertensive, cyclooxygenase inhibitor, or opioid action. Thus, stable cell lines can be suitable for expression signature-based screening of a diverse range of activities. A SOGS-based system also discriminates physiologically active from inactive compounds, identifies drugs with off-target side effects, and incorporates a quantitative method for assigning confidence to individual predictions that, at its most stringent, approaches 100% accuracy. The capacity to resolve multiple distinct drug activities while simultaneously discriminating inactive and potential false-positive compounds in a cell line presents a unified framework for streamlined chemical genomic drug discovery.

The use of phospholipid fatty acid analysis to measure impact of acid rock drainage on microbial communities in sediments.

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0-0.1 km), compared with sites further downstream (2.5-18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments' metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:2omega6; the sulfate-reducing bacteria marker 10Me16:1omega7; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.

Differential tramadol and O-desmethyl metabolite levels in brain vs. plasma of mice and rats administered tramadol hydrochloride orally.

OBJECTIVE: To investigate a possible differential brain uptake of tramadol vs. its major metabolite (O-desmethyl tramadol; M1) in mice and rats. METHODS: An extraction and measurement technique (gas chromatograph equipped with a nitrogen phosphorus detector) was used to measure plasma and brain levels of tramadol and M1 at intervals 10-300 min after oral dosing of tramadol hydrochloride to mice and rats. RESULTS: For all doses of tramadol administered (5, 10, 20, or 40 mg/kg), tramadol and M1 plasma levels were greatest 10 min after dosing: in mice, peak tramadol plasma levels were 47.75-736.72 ng/mL and peak M1 levels were 75.30-1084.92 ng/mL; in rats, peak tramadol plasma levels were 185.03-455.81 ng/mL and peak M1 levels were 106.74-455.70 ng/mL. Tramadol brain levels were also greatest 10 min after dosing. In mice, peak tramadol brain levels were 226.42-1847.46 ng/g. Peak M1 levels (72.17-572.97 ng/g) occurred 20-60 min after dosing. In rats, peak tramadol brain levels were 258.50-1777.37 ng/g and peak M1 levels were 80.35-289.60 ng/g. In mice, the ratio of tramadol/M1 in plasma was 0.5-1.0 throughout the measurements, whereas the ratio in brain was about 10 at 10 min and about 2 from 20 to 50 min. In rats, the ratio of tramadol/M1 in plasma was 0.5-1.5, whereas the ratio in brain was about 15 at 10 min and about 4-7 thereafter. CONCLUSION: In mice and rats, there appears to be preferential brain vs. plasma distribution of tramadol over M1.

Effects of pre- and postnatal corticosterone exposure on the rat hippocampal GABA system.

Several lines of evidence have implicated prenatal stress and the hippocampal GABA system in the pathophysiology of schizophrenia, and prenatal stress is believed to increase the risk for schizophrenia through alterations of this neurotransmitter. To explore this hypothesis, we treated male rats pre- and/or postnatally (P48 and P60) with either corticosterone (CORT) or vehicle to establish three study groups: VVV, receiving vehicle at all three time points; VCC, receiving vehicle prenatally and CORT at both postnatal timepoints; and CCC, receiving CORT at all three timepoints. Animals were sacrificed at either 24 h or 5 days after final injection and examined for mRNA levels of GAD65, GAD67, and the GABA(A) receptor subunits alpha2 and gamma2. At 24 h, GAD65 mRNA was decreased in CA1, CA2, CA4, and dentate gyrus (DG) of VCC rats; this effect was either decreased or reversed in CCC-treated animals. No effect was detected in GAD67 mRNA at 24 h. At 5 days, CORT treatment increased GAD67 mRNA levels in CA1, CA3, and DG. Prenatal treatment with CORT was associated with increased responsiveness only in CA3 and DG. For the GABAA receptor, alpha2 subunit mRNA did not show any change in response to CORT treatment, while that for the gamma2 subunit was decreased in CA2 of both VCC- and CCC-treated animals. Consistent with gamma2 subunit mRNA decreases, benzodiazepine (BZ) receptor binding activity was decreased in CA2 with CORT treatment. Prenatal CORT exposure neither increased nor decreased this effect. These results demonstrate that CORT administration is associated with a complex regulation of mRNA expression for pre- and postnatal aspects of the hippocampal GABA system. Under these conditions, prenatal exposure to CORT may sensitize some of these effects, but does not fundamentally alter the nature of this response.

Gas chromatographic method using nitrogen-phosphorus detection for the measurement of tramadol and its O-desmethyl metabolite in plasma and brain tissue of mice and rats.

A method that allows the measurement of plasma and brain levels of the centrally-acting analgesic tramadol and its major metabolite (O-desmethyl tramadol) in mice and rats was developed using gas chromatography equipped with nitrogen-phosphorus detection (GC-NPD). Plasma samples were extracted with methyl tert.-butyl ether (MTBE) and were injected directly into the GC system. Brain tissue homogenates were precipitated with methanol, the resulting supernatant was dried then acidified with hydrochloric acid. The aqueous solution was washed with MTBE twice, alkalinized, and extracted with MTBE. The MTBE layer was dried, reconstituted and injected into the GC system. The GC assay used a DB-1 capillary column with an oven temperature ramp (135 to 179 degrees C at 4 degrees C/min). Dextromethorphan was used as the internal standard. The calibration curves for tramadol and O-desmethyl tramadol in plasma and brain tissue were linear in the range of 10 to 10000 ng/ml (plasma) and ng/g (brain). Assay accuracy and precision of back calculated standards were within +/- 15%.

Unexpected and pronounced antinociceptive synergy between spinal acetaminophen (paracetamol) and phentolamine.

Acetaminophen was administered to mice by spinal (intrathecal, i.t.) injection alone or with phentolamine (11.3 microg = 0.03 micromol). Acetaminophen produced dose-related antinociception in the abdominal irritant test with an ED(50) value of 137.2 microg (0.9 micromol) Phentolamine had no effect. For combined administration, the potency of acetaminophen was significantly increased (ED50=24.4 vs. 137.2 microg), indicative of multiplicative interaction and strong synergism. These results reveal the significant and surprising interaction of spinal cord adrenoceptors or ion channel subtypes with acetaminophen-induced antinociception.

Discovery of "self-synergistic" spinal/supraspinal antinociception produced by acetaminophen (paracetamol).

The mechanism of the analgesic action of one of the world's most widely used drugs-acetaminophen (paracetamol)-remains largely unknown more than 100 years after its original synthesis. Based on the present findings, this elusiveness appears to have resulted from experimental strategies that concentrated on a single target site or mechanism. Here we report on the use of analyses that we previously developed to investigate possible brain/spinal-cord site-site interaction in acetaminophen-induced antinociception. Spinal (intrathecal) administration of acetaminophen to mice produced dose-related, naloxone-insensitive antinociception with an ED(50) value of 137 (S.E. = 23) microgram = 907 (S.E. =153) nmol. In contrast, supraspinal (i.c.v.) acetaminophen administration had no effect. However, combined administration of acetaminophen in fixed ratios to brain and spinal cord produced synergistic antinociception, ED(50) = 57 (S.E. = 9) microgram, that reverted toward additivity, ED(50) = 129 (S.E. = 23) microgram, when the opioid antagonist naloxone was given spinally (3.6 microgram = 10 nmol) or s.c. (3.6 mg/kg). These findings demonstrate for the first time that acetaminophen-induced antinociception involves a "self-synergistic" interaction between spinal and supraspinal sites and, furthermore, that the self-synergy involves an endogenous opioid pathway.

Effects of age on gene expression during estrogen-induced synaptic sprouting in the female rat.

Age and estrogen treatment influenced fiber outgrowth and compensatory neuronal sprouting after unilateral entorhinal cortex lesions (ECL) which model Alzheimer disease-like deafferentation in the dentate gyrus of the hippocampus. In young F344 rats (3 months old), ovariectomy (OVX) decreased reactive fiber outgrowth by 60%. Sprouting in middle-aged rats (18 months old) was reduced in intact females; no further reduction was caused by OVX. Several astrocyte mRNAs were measured in the dentate gyrus of young and middle-aged female rats in three different estrogen states (sham OVX, OVX, or OVX + estradiol) 1 week after ECL. Glial fibrillary acidic protein (GFAP) mRNA was twofold greater in middle-aged rats than young, although both ages showed threefold increases in response to ECL. In prior studies GFAP was found to be decreased by estradiol treatment 3-4 days after ECL; in this study GFAP mRNA had returned to sham OVX levels in young rats by 7 days post-ECL. Surprisingly, estradiol treatment increased GFAP mRNA levels by 25% above OVX in middle-aged rats. Apolipoprotein E (apoE) mRNA was decreased 20% by age in the dentate, although both age groups showed a 25% increase in apoE mRNA in response to ECL. Apolipoprotein J (apoJ) mRNA was increased 20% in the dentate gyrus of middle-aged rats, and both age groups responded to ECL with a 65% increase in apoJ mRNA. The estrogen state did not alter levels of either apolipoprotein mRNA in the deafferented dentate. The data suggest that the estrogen-induced decrease of GFAP in response to lesions does not persist at 7 days post-ECL during sprouting. Overall effects of age on the dentate gyrus include elevated GFAP mRNA and decreased apoE mRNA. The cortical wound site showed consistent enhancement of GFAP mRNA in both age groups by estradiol above sham OVX and greater responses in middle-aged rats.

Determination of the adsorption of tramadol hydrochloride by activated charcoal in vitro and in vivo.

Although tramadol is one of the most widely used centrally acting analgesics worldwide, no literature is available regarding adsorption of tramadol HCl powder or tablets (Ultram; 50 mg tramadol HCl per tablet) by activated charcoal (AC) for use as potential adjunct treatment of overdose. The present study incorporated a novel combination of in vitro and in vivo methods to investigate this question. Based on a binding curve of tramadol UV absorbance (UV(a); 225 nm) plotted against the amount of AC, the ratio of amount of tramadol completely adsorbed by AC was 0.05 mg/mg. Also based on UV(a), no tramadol was detected in filtrate of slurries in which up to 62 tablets of Ultram were mixed with 50 g AC; 4.6% of unbound tramadol was detected when 100 tablets of Ultram were mixed with AC. The ratio of amount of tramadol completely adsorbed by AC in this test was 0.10. In vivo, co-administration of 0.1 g/ml of AC produced a 13- to 14-fold rightward shift in tramadol's antinociceptive dose-response curve and a 1.6-fold rightward shift in tramadol's lethality dose-response curve.

Process-based pharmacology in neuroanesthesia.

The present review focuses on the process by which selected pharmacologic agents can be employed in the management of specific problems that arise during surgical procedures, including tumor or trauma with elevated intracranial pressure, previously ruptured aneurysm, and procedures that may require some degree of controlled hypertension, such as carotid endarterectomy or temporary clipping. A balanced view between older established data, newer information, and long-term clinical practice in caring for such patients is presented. The emphasis is on intravenous rather than inhaled agents; issues that involve neuromuscular blockers are not addressed here.

Localization of cells preferentially expressing GAD(67) with negligible GAD(65) transcripts in the rat hippocampus. A double in situ hybridization study.

Two major forms of glutamic acid decarboxylase (GAD) are present in the mammalian brain, a 65-kDa isoform (GAD(65)) and a 67-kDa isoform (GAD(67)), and it is usually assumed that all GABAergic neurons contain both. The two forms have not yet been colocalized to the same neurons, because the GAD(65) protein is found almost exclusively in axon terminals, while GAD(67) is found predominantly in the cell body. Using double in situ hybridization (DISH) with both radioactive [35S] and non-radioactive (digoxigenin, DIG) probes, the distributions of GAD(65) and GAD(67) mRNA have been simultaneously examined in the rat hippocampus. The results suggest that [35S] radioprobes are slightly more sensitive than DIG probes, and that the reversal of labels is necessary in DISH studies to determine whether a neuronal subtype which expresses only one isoform of GAD may be present. The data indicate that the majority of cells (90%) showing labeling were labeled for both GAD(65) and GAD(67) mRNA. In sectors CA1 and CA3 approximately 5-10% of the cells positive for GAD(67) showed little or no detectable GAD(65) mRNA. In the hilus, however, GAD(65) levels were higher, and all cells seem to express both GAD(65) and GAD(67) mRNA. Taken together, these results support the view that most GABAergic neurons in the hippocampus express both GAD(65) and GAD(67). However, it appears that some interneurons in the CA subfields differ from "classic" GABAergic interneurons by preferentially expressing the 67-kDa isoform of GAD under baseline conditions, with GAD(65) mRNA levels very low or absent.

Variations of synaptotagmin I, synaptotagmin IV, and synaptophysin mRNA levels in rat hippocampus during the estrous cycle.

Periodic changes in ovarian steroid levels during fertility cycles affect learning both in humans and in rats in parallel with electrophysiological and morphological fluctuations in selective neuronal populations. In particular, during the estrous cycle of the female rat, hippocampal CA1 region undergoes cyclic modifications in synaptic density. To investigate the molecular mechanisms involved in synaptic remodeling during the estrous cycle, we analyzed the expression of three presynaptic markers, synaptotagmin I, synaptotagmin IV, and synaptophysin, in the female adult rat brain by in situ hybridization. Relative abundance in mRNA for these three markers was quantified at four phases of the estrous cycle: diestrus, proestrus (AM and PM), and estrus. mRNA levels for syt1 exhibited cyclic variations in pyramidal neurons of the CA3 region of hippocampus during the estrous cycle, while mRNA levels for syt4 and SYN were relatively invariant in this or other regions of the hippocampus. Because CA3 pyramidal neurons make synaptic contacts in CA1, modulation of syt1 expression in CA3 may participate in the changes in synaptic density observed in CA1 during the estrous cycle. Furthermore, both syt1 and SYN mRNA varied cyclically in layer II, but not in layer III of entorhinal cortex, while syt4 remained unchanged throughout the cycle. These data suggest that regular variations in steroid hormone levels during fertility cycles may alter the properties of several networks involved in information processing and learning and memory through altered levels of presynaptic proteins.

Differential cholera-toxin sensitivity of supraspinal antinociception induced by the cannabinoid agonists delta9-THC, WIN 55,212-2 and anandamide in mice.

Intracerebroventricular (i.c.v.) administration to mice of delta9-tetrahydrocannabinol (delta9-THC), WIN 55,212-2 or the endogenous cannabinoid anandamide induced dose-related antinociception in the 55 degrees C warm-water tail-flick test. Pretreatment (24 h, i.c.v.) with pertussis toxin dose-dependently reduced the antinociceptive effect of delta9-THC (955 nmol), WIN 55,212-2 (30 nmol) and anandamide (135 nmol) (IC50 = 0.13, 5.5, and 0.32 nmol, respectively). In contrast, pretreatment (24 h, i.c.v.) with cholera toxin (0.1-3.0 mg) reduced the antinociception of WIN 55,212-2, had minimal effect on delta9-THC, and dose-dependently increased the antinociception of anandamide (ED50 = 0.50 nmol). These data suggest differences in the receptor-effector coupling of delta9-THC, WIN 55,212-2 and anandamide in supraspinal-induced antinociception in mice.

Response surface analysis of synergism between morphine and clonidine.

Graded doses of morphine sulfate and clonidine hydrochloride were administered intrathecally to mice that were then tested for antinociception in the 55 degrees C tail immersion test. The dose-effect relations of each compound were used in calculations that permitted the construction of a three-dimensional plot of the expected additive effect (vertical scale) against the planar domain of dose pairs representing combinations administered simultaneously. This additive response surface became the reference surface for viewing the actual effects produced by three different fixed-ratio combinations of the drugs that were used in our tests. Each combination produced effects significantly greater than indicated by the additive surface, thereby illustrating marked synergism and a method for quantifying the synergism. This quantification, measured by the value of the interaction index (alpha), was found to be dependent on the fixed-ratio combination; accordingly, the actual response surface could not be described by a single value of the index alpha. Furthermore, we found that application of the common method of isoboles gave estimates of the index that agreed well with those obtained from the more extensive surface analysis. These results confirm earlier studies, which found synergism for these drugs while also providing surface views of additivity and synergism that form the basis of isobolographic analysis.

Mu receptor and Gi2alpha antisense attenuate [D-Met2]-FMRFamide antinociception in mice.

FMRFamide (Phe-Met-Arg-Phe-NH2) and several analogs produce centrally-mediated, naloxone-reversible antinociception, but have minimal affinity for opioid receptor (sub)types. In the present study, the antinociception in mice (55 degrees C tail-flick test) produced by supraspinal (intracerebroventricular; i.c.v.) administration of [D-Met2]-FMRFamide (a stable analog of FMRFamide) was attenuated by pretreatment with i.c.v. oligodeoxyribonucleotide antisense to the opioid mu receptor or by antisense to the Gi2alpha G-protein subunit. These data suggest that [D-Met2]-FMRFamide produces its antinociception via an opioid interneuron.