MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

Development of an on-animal separation-based sensor for monitoring drug metabolism in freely roaming sheep.

The development of an on-animal separation-based sensor that can be employed for monitoring drug metabolism in a freely roaming sheep is described. The system consists of microdialysis sampling coupled to microchip electrophoresis with electrochemical detection (MD-ME-EC). Separations were accomplished using an all-glass chip with integrated platinum working and reference electrodes. Discrete samples from the microdialysis flow were introduced into the electrophoresis chip using a flow-gated injection approach. Electrochemical detection was accomplished in-channel using a two-electrode isolated potentiostat. Nitrite was separated by microchip electrophoresis using reverse polarity and a run buffer consisting of 50 mM phosphate at pH 7.4. The entire system was under telemetry control. The system was first tested with rats to monitor the production of nitrite following perfusion of nitroglycerin into the subdermal tissue using a linear probe. The data acquired using the on-line MD-ME-EC system were compared to those obtained by off-line analysis using liquid chromatography with electrochemical detection (LC-EC), using a second microdialysis probe implanted parallel to the first probe in the same animal. The MD-ME-EC device was then used on-animal to monitor the subdermal metabolism of nitroglycerin in sheep. The ultimate goal is to use this device to simultaneously monitor drug metabolism and behavior in a freely roaming animal.

Microperfusion of 3-MPA into the brain augments GABA.

In vivo effects of microperfusion of a GABA synthesis inhibitor (3-MPA) into the striatum and hippocampus on amino acid concentrations and electrical neuronal activity were investigated. Paradoxical elevations in GABA in the striatum (5-fold in anesthetized and 50-fold in awake rats) and hippocampus (2-fold in anesthetized and 15-fold in awake rats) were documented under steady-state concentrations of 3-MPA along with expected increases in glutamate (a 15-fold increase and a 250-fold increase in the striatum of anesthetized and awake rats, respectively; a 7-fold increase and a 25-fold increase in the hippocampus of anesthetized and awake rats, respectively). There was no clear epileptiform or seizure activity. Explanations for the paradoxical increase in GABA are offered, and emphasis is placed on the dependency of disinhibition on the model in which its effects are studied as well as on the prevailing level of activation of the probed network.

A parallel dual-electrode detector for capillary electrophoresis.

An approach to on-capillary dual-electrode detection for CE using a parallel electrode configuration has been developed. The parallel configuration provides two operating modes. In the first mode, one working electrode is held at an oxidizing potential and the second working electrode is held at a reducing potential. This results in redox cycling of analytes between the oxidized and reduced forms, enhancing sensitivity compared to single-electrode detection. In the second mode, both working electrodes are held at different oxidizing potentials. This mode provides electrochemical characterization of electrophoretic peaks. In the redox cyclying mode, signal enhancement of up to twofold was observed for the dual-electrode detection of phenolic acid standards compared to single-electrode detection. Variation in response of less than 10% from electrode to electrode was determined (at a concentration of 60 nM) indicating reproducible fabrication. LODs were determined to be as low as 5.0 nM for dual-electrode configuration. Using the dual-potential mode peak identification of targeted phenolic acids in whiskey samples were confirmed based on both migration time and current ratios.

Determination of GABA, glutamate and carbamathione in brain microdialysis samples by capillary electrophoresis with fluorescence detection.

Disulfiram has been used as a deterrent in the treatment of alcohol abuse for almost 60 years. Our laboratory has shown that a disulfiram metabolite, S-(N,N-diethylcarbamoyl) glutathione (carbamathione), is formed from disulfiram and appears in the brain after the administration of disulfiram. Carbamathione does not inhibit aldehyde dehydrogenase but has been shown to be a partial non-competitive inhibitor of the N-methyl-D-aspartic acid glutamate (Glu) receptor. In light of disulfiram's apparent clinical effectiveness in cocaine dependence, and carbamathione's effect on the N-methyl-D-aspartic acid receptor, the effect of carbamathione on brain Glu and γ-aminobutyric acid (GABA) needs to be further examined. A CE-LIF method based on derivatization with napthalene-2,3-dicarboxyaldehyde to simultaneously detect both neurotransmitter amino acids and carbamathione in brain microdialysis samples is described. The separation of Glu, GABA and carbamathione was carried out using a 50 mmol/L boric acid buffer (pH 9.6) on a 75 cm×50 µm id fused-silica capillary (60 cm effective) at +27.5 kV voltage with a run time of 11 min. The detection limits for Glu, GABA and carbamathione were 6, 10 and 15 nmol/L, respectively. This method was used to monitor carbamathione and the amino acid neurotransmitters in brain microdialysis samples from the nucleus accumbens after the administration of an intravenous dose of the drug (200 mg/kg) and revealed a carbamathione-induced change in GABA and Glu levels. This method demonstrates a simple, rapid and accurate measurement of two amino acid neurotransmitters and carbamathione for in vivo monitoring in the brain using microdialysis sampling.

Neurochemical investigation of multiple locally induced seizures using microdialysis sampling: Epilepsy effects on glutamate release.

The objective of this study was to develop an in vivo model for locally induced epilepsy. Epilepsy is a prominent neurological disorder that affects millions of people worldwide. Patients may experience either global seizures, affecting the entire brain, or focal seizures, affecting only one brain region. The majority of epileptic patients experience focal seizures but they go undiagnosed because such seizures can be difficult to detect. To better understand the effects of focal epilepsy on the neurochemistry of a brain region with high seizure diathesis, an animal model for locally induced seizures in the hippocampus was developed. In this model, two seizure events were chemically induced by administering the epileptogenic agent, 3-mercaptopropionic acid (3-MPA), to the hippocampus to disturb the balance between excitatory and inhibitory neurotransmitters in the brain. Microdialysis was used for local delivery of 3-MPA as well as for collection of dialysate for neurochemical analyses. Two periods of seizures separated by varying inter-seizure recovery times were employed, and changes in the release of the excitatory transmitter, glutamate, were measured. Significant differences in glutamate release were observed between the first and second seizure episodes. Diminished glutamate biosynthesis, enhanced glutamate re-uptake, and/or neuronal death were considered possible causes of the attenuated glutamate release during the second seizure episode. Biochemical measurements were indicative that a combination of these factors led to the attenuation in glutamate release.

CE-LIF method for the separation of anthracyclines: application to protein binding analysis in plasma using ultrafiltration.

Anthracyclines are chemotherapeutic drugs that are widely used in the treatment of cancers such as lung and ovarian cancers. The simultaneous determination of the anthracyclines, daunorubicin, doxorubicin and epirubicin, was achieved using CE coupled to LIF, with an excitation and emission wavelength of 488 and 560 nm, respectively. Using a borate buffer (105 mM, pH 9.0) and 30% MeOH, a stable and reproducible separation of the three anthracyclines was obtained. The method developed was shown to be capable of monitoring the therapeutic concentrations (50-50 000 ng/mL) of anthracyclines. LODs of 10 ng/mL, calculated at an S/N = 3, were achieved. Using the CE method developed, the in vitro protein binding to plasma was measured by ultrafiltration, and from this investigation the estimated protein binding was determined to be in the range of 77-94%.

The direct comparison of health and ulcerated stomach tissue: a multiple probe microdialysis sampling approach.

The ability to directly compare gastric ulcerated and healthy tissue would aid in the understanding of the physiological differences between these tissue types. Presently, these comparisons can only be drawn by the use of separate animal groups, which results in increased study variability. The focus of this research was to develop a four-probe microdialysis sampling approach to directly compare ulcerated and healthy tissue in the same animal. After controlled chemical ulcer induction, probes were implanted in the ulcerated and healthy stomach submucosa, stomach lumen and in the blood. To assess the significance of this multiple probe approach, drug concentrations in each probe location were monitored after selected compounds were dosed to the ligated stomach by oral gavage. Analysis of the dialysate samples was performed by HPLC-UV and concentration-time curves and pharmacokinetics analyses were used to determine differences between these tissue types.

The development of multiple probe microdialysis sampling in the stomach.

A multiple probe approach of implanting microdialysis probes into each separate tissue layer would better represent sampling from the stomach. Presently, microdialysis sampling experiments are performed with only a single probe in the submucosa to represent sampling from the stomach tissue. The focus of this research was to develop a four-probe microdialysis sampling design to simultaneously monitor the stomach lumen, mucosa, submucosa and in the blood of the rat. Due to the small outer diameter of the microdialysis probe (350 microm), implantation into each separate layer was achieved with confirmation of probe location from histological examination. To assess the significance of sampling by this approach, multiple probe microdialysis sampling was used to monitor drug absorption in the stomach. Salicylic acid, caffeine and metoprolol were individually dosed to the ligated stomach. Analysis of the dialysate samples was performed by HPLC-UV and concentration-time curves and pharmacokinetics analysis were used to determine differences between the different probe locations.

Development of tissue-targeted metabonomics. Part 1. Analytical considerations.

Tissue-targeted metabonomics provides tissue specific metabolic information while still retaining the profiling approach of traditional metabonomics. Microdialysis sampling is used to generate site-specific samples of endogenous metabolites. The dialysate samples are subjected to proton NMR analysis with data analysis by principal components analysis and partial least squares regression. In this study, sample and data pretreatment methods were examined for their impact on the quality of the data analysis. Specifically, the effects of speed vacuuming, sample solubility, sample pH stability, and sample storage stability were examined. Data pretreatment methods examined included the effects of standardization and normalization to internal standards. In addition, the ability of tissue-targeted metabonomics to generate time trend data was explored and more fully characterized using principal components analysis and partial least squares regression.

An investigation into the pharmacokinetics of 3-mercaptopropionic acid and development of a steady-state chemical seizure model using in vivo microdialysis and electrophysiological monitoring.

OBJECTIVES: The goal of the present study was to develop a chemical seizure model using the convulsant, 3-mercaptopropionic acid (3-MPA). A pharmacodynamics approach was taken, combining in vivo microdialysis sampling with electrophysiological methods to simultaneously monitor, in real-time, the 3-MPA concentration in the brain and the corresponding electrocorticographic (ECoG) activity. METHODS: The 3-MPA was administered in two doses (50 and 100 mg/kg) in order to study its pharmacokinetics. Microdialysis samples were collected from the striatum, hippocampus, and jugular vein every 5 min. The microdialysates were analyzed using high-performance liquid chromatography with electrochemical detection (HPLC-EC). The ECoG activity was monitored via screws placed onto the cortex. Noncompartmental pharmacokinetics analysis was performed to obtain the elimination constants (K(e)), the maximum concentration (C(max)), the time to achieve maximum concentration (T(max)), and the area under the concentration-time curves (AUC(inf)). RESULTS: The average brain K(e) for the 50 and the 100mg/kg doses were 0.060 and 0.018 min(-1), respectively. The brain AUC(inf) for the 50 and 100mg/kg doses were 353 and 2168 mg min(-1)mL(-1), respectively. This led to a 67-fold increase in the observed number of seizures in the higher dose with the average seizure intensity double that of the smaller dose. These data led to the dosing scheme for the chemical seizure model of administering a 3-MPA loading dose of 60 mg/kg followed by a constant infusion of 50 mg/(kg min(-1)). CONCLUSIONS: This study describes, to our knowledge, the first successful attempt to combine in vivo microdialysis with electrophysiology to monitor in real-time, the concentration and effects of 3-MPA in the brain. This led to the development of a steady-state chemical seizure model.

Application of a column selection system and DryLab software for high-performance liquid chromatography method development.

This paper describes a strategy for the development of chromatographic methods for drug candidates based upon the use of simple MS compatible mobile phases and optimization of the chromatographic selectivity through variations of the stationary phase and mobile phase pH. The strategy employs an automated column selection system and a series of HPLC columns, varying in hydrophobicity and silanol activity, in combination with DryLab software to develop chromatographic methods for the separation of mixtures of bupivacaine and its metabolites; acidic, basic, and neutral compounds; and atenolol, nitrendipine, and their degradation products.

A Capillary Electrophoresis Electrospray Ionization-Mass Spectrometry Method using a Borate Background Electrolyte for the Fingerprinting Analysis of Flavonoids in Ginkgo biloba Herbal Supplements.

A laboratory-built sheath liquid capillary electrophoresis-mass spectrometry interface was used to develop a qualitative method for fingerprinting analysis of 14 structurally similar flavones, flavonols, flavonones, and several representative glycosides in plant samples. The migration order of the flavonoids was dependent on a the number of hydroxyl groups present on the flavonoid B-ring, extent of conjugation, number of glycosidic functionalities, and ability of the flavonoid to form stable borate complexes with the background electrolyte. Parent ion scans of the flavonoids yielded [M-H]-, except for catechol containing flavonoids, which were detected as borate adducts. These adducts can be used diagnostically to determine the presence or absence of catechol groups on unknown polyphenolic compounds. Product ion scans of the flavonoid glycosides and borate adducts typically yielded the deprotonated aglycone fragment as the base peak, which could be used to confirm the base structure of the flavonoid. This method's utility was demonstrated by analyzing flavonoids present in ethanolic extracts of Ginkgo biloba herbal supplements.

On-column preconcentration of glutathione and glutathione disulfide using pH-mediated base stacking for the analysis of microdialysis samples by capillary electrophoresis.

Capillary electrophoresis (CE) has become a useful analytical tool for the analysis of microdialysis samples. However, CE with UV detection (CE-UV) does not provide detection limits sufficient to quantify glutathione (GSH) and glutathione disulfide (GSSG) in biological samples such as liver microdialysates, because of the small optical path length in the capillary. To overcome this limitation, an on-column preconcentration technique, pH-mediated base stacking, was used in this study to improve the sensitivity of CE-UV. This stacking technique allowed large volumes of high ionic strength sample injection without deterioration of the separation efficiency and resolution. A 26-fold increase in sensitivity was achieved for both GSH and GSSG using the pH-mediated base stacking, relative to normal injection without stacking. The limit of detection for GSH and GSSG was found to be 0.75 microM (S/N=6) and 0.25 microM (S/N=6), respectively. The developed method was used to analyze GSH and GSSG in liver microdialysates of anesthetized Sprague Dawley male rats. The basal concentrations of GSH and GSSG in the liver microdialysates of male rats were found to be 4.73+/-2.08 microM (n=7) and 5.52+/-3.66 microM (n=7), respectively.

Determination of 8-oxoguanine and 8-hydroxy-2'-deoxyguanosine in the rat cerebral cortex using microdialysis sampling and capillary electrophoresis with electrochemical detection.

A rapid and sensitive method to determine 8-oxoguanine (8oxoG) and 8-hydroxydeoxyguanosine (8OHdG), biomarkers for oxidative DNA damage, in cerebral cortex microdialysate samples using capillary electrophoresis (CE) with electrochemical detection (CEEC) was developed. Samples were concentrated on-column using pH-mediated stacking for anions. On-column anodic detection was performed with a carbon fiber working electrode and laser-etched decoupler. The method is linear over the expected extracellular concentration range for 8oxoG and 8-OHdG during induced ischemia-reperfusion, with R.S.D. values

Tissue targeted metabonomics: metabolic profiling by microdialysis sampling and microcoil NMR.

The concentration of low molecular weight compounds in tissues can yield valuable information about the metabolic state of an organism. Studies of changes in the metabolic state or metabonomics can reflect disease pathways, drug action, or toxicity. This research aims to develop a new approach, tissue targeted metabonomics. Microdialysis sampling and microcoil NMR analysis are employed to compare basal and ischemic metabolic states of various tissues (blood, brain, and heart) of Sprague-Dawley rats. Microdialysis sampling is localized, making the metabolic profile tissue specific. Coupling to NMR analysis is highly advantageous, because a complete metabolic profile is obtained in a single spectrum. However, small sample volumes and low analyte concentrations make analysis of microdialysis samples challenging. Microcoil NMR uses low sample volumes and has improved mass sensitivity, relative to standard 5 mm probes. The coupling of these techniques is a potentially powerful tool for metabonomics analysis.

N-acetyl-S-(N,N-diethylcarbamoyl) cysteine in rat nucleus accumbens, medial prefrontal cortex, and in rat and human plasma after disulfiram administration.

Disulfiram (DSF), a treatment for alcohol use disorders, has shown some clinical effectiveness in treating addiction to cocaine, nicotine, and pathological gambling. The mechanism of action of DSF for treating these addictions is unclear but it is unlikely to involve the inhibition of liver aldehyde dehydrogenase (ALDH2). DSF is a pro-drug and forms a number of metabolites, one of which is N-acetyl-S-(N,N-diethylcarbamoyl) cysteine (DETC-NAC). Here we describe a LCMS/MS method on a QQQ type instrument to quantify DETC-NAC in plasma and intracellular fluid from mammalian brain. An internal standard, the N,N-di-isopropylcarbamoyl homolog (MIM: 291>128) is easily separable from DETC-NAC (MIM: 263>100) on C18 RP media with a methanol gradient. The method's linear range is 0.5-500 nM from plasma and dialysate salt solution with all precisions better than 10% RSD. DETC-NAC and internal standards were recovered at better than 95% from all matrices, perchloric acid precipitation (plasma) or formic acid addition (salt) and is stable in plasma or salt at low pH for up to 24 h. Stability is observed through three freeze-thaw cycles per day for 7 days. No HPLC peak area matrix effect was greater than 10%. A human plasma sample from a prior analysis for S-(N,N-diethylcarbamoyl) glutathione (CARB) was found to have DETC NAC as well. In other human plasma samples from 62.5 mg/d and 250 mg/d dosing, CARB concentration peaks at 0.3 and 4 nM at 3 h followed by DETC-NAC peaks of 11 and 70 nM 2 h later. Employing microdialysis sampling, DETC-NAC levels in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and plasma of rats treated with DSF reached 1.1, 2.5 and 80 nM at 6h. The correlation between the appearance and long duration of DETC-NAC concentration in rat brain and the persistence of DSF-induced changes in neurotransmitters observed by Faiman et al. (Neuropharmacology, 2013, 75C, 95-105) is discussed.

Determination of cannabinoids in hair using high-pH* non-aqueous electrolytes and electrochemical detection. Some aspects of sensitivity and selectivity.

Non-aqueous capillary electrophoresis with electrochemical detection (NACE-ED) was applied to the determination of cannabinoids in hair. The effect of different electrolyte compositions on the selectivity of the separation of tetrahydrocannabinol (THC), cannabinol (CBN), cannabidiol (CBD) and tetrahydrocannabinol carboxylic acid (THCA) was studied. Complete electrophoretic resolution was obtained using a strongly basic background electrolyte consisting of 5 mM sodium hydroxide dissolved in acetonitrile-methanol (1:1). Electrochemical detection yielded well defined signals in the oxidation mode. In order to obtain low limits of detection experimental parameters, which determine the sensitivity and the noise level, were optimized. A crucial parameter for sensitive measurements using a wall-tube flow cell as end-column detector is the distance between the capillary outlet and the working electrode. The highest signal-to-noise ratio using a 50 microm I.D. capillary was obtained at a distance of 25 microm. When the capillary outlet was moved away from the working electrode, thus reducing the strength of the separation field present at the working electrode, a large low frequency noise developed. This rise was attributed to disturbances of the hydrodynamic pattern in the flow cell. Analytical aspects such as sensitivity, reproducibility and selectivity were addressed in this work. The precision of NACE-ED regarding migration time and peak height for a sample containing 1 microg/ml THC was 0.4% and 1.1% (RSD), respectively (n=5). The calibration curve was linear for concentrations ranging between 0.1 and 10 microg/ml (r=0.998). The limit of detection for THC was 37 ng/ml, which is almost two orders of magnitude lower when compared with on-column UV detection. The method was evaluated using hair samples containing cannabinoids as sample material.

Application of capillary electrophoresis with pH-mediated sample stacking to analysis of coumarin metabolites in microsomal incubations.

A sensitive method for the analysis of metabolites of coumarin by capillary electrophoresis (CE), incorporating pH-mediated sample stacking, was developed. The analytes were detected in phosphate buffer (pH 7.5; 25 mM), the matrix of the microsomal incubations. Detection was by direct UV absorbance. The three metabolites studied were 7-hydroxycoumarin (7-OHC), 4-hydroxycoumarin (4-OHC) and 2-hydroxyphenylacetic acid (HPAA), and the limits of detection of the analytes were 0.1, 0.5 and 0.3 microM, respectively. The developed method was then applied to microsomal incubations of coumarin. Male Cynomologus monkey microsomes were used in the study and 7-OHC was detected in the incubation mixture.

Correlation of 3-mercaptopropionic acid induced seizures and changes in striatal neurotransmitters monitored by microdialysis.

OBJECTIVES: The goal of this study was to use a status epilepticus steady-state chemical model in rats using the convulsant, 3-mercaptopropionic acid (3-MPA), and to compare the changes in striatal neurotransmission on a slow (5min) and fast (60s) timescale. In vivo microdialysis was combined with electrophysiological methods in order to provide a complete evaluation of the dynamics of the results obtained. OBJECTIVE: To compare the effects of a steady-state chemical model pof status epilepticus on striatal amino-acid and amine neurotransmitters contents, as measured via in vivo microdialysis combined with electrophysiological methods. Measurements were performed on samples collected every 60s and every 5min. "Fast" (60s) and "slow" (5min) sampling timescales were selected, to gain more insight into the dynamics of GABA synthesis inhibition and of its effects on other neurotransmitters and on cortical electrical activity. METHODS: 3-MPA was administered in the form of an intra-venous load (60mg/kg) followed by a constant infusion (50mg/kg/min) for min. Microdialysis samples were collected from the striatum at intervals of 5min and 60s and analyzed for biogenic amine and amino acid neurotransmitters. ECoG activity was monitored via screws placed over the cortex. RESULTS: In the 5min samples, glutamate (Glu) increased and γ-aminobutyric acid (GABA) decreased monotonically while changes in dopamine (DA) concentration were bimodal. In the sixty second samples, Glu changes were bimodal, a feature that was not apparent with the 5min samples. ECoG activity was indicative of status epilepticus. CONCLUSIONS: This study describes the combination of in vivo microdialysis with electrophysiology to monitor the effect of 3-MPA on neurotransmission in the brain. This led to a better understanding of the chemical changes in the striatum due to the applied 3-MPA chemical model of status epilepticus.