Paul Daenens

Effect of fluoxetine on a neuronal, voltage-dependent potassium channel (Kv1.1)

1 Fluoxetine (Prozac) is widely used as an antidepressant drug and is assumed to be a selective 5‐hydroxytryptamine (5‐HT) reuptake inhibitor (SSRI). Claims that its beneficial psychotropic effects extend beyond those in treatment of depression have drawn clinical and popular attention to this compound, raising the question of whether there is anything exceptional about the supposed selective actions. 2 We have used the voltage clamp technique to study the effect of fluoxetine on a neuronal, voltage‐dependent potassium (K+) channel (RCK1; Kv1.1), expressed in Xenopus laevis oocytes. This channel subunit is abundantly expressed in the central nervous system and K+ channels containing this subunit are involved in the repolarization process of many types of neurones. 3 Blockade of the K+ currents by fluoxetine was found to be use‐ and dose‐dependent. Wash‐out of this compound could not be achieved. Fluoxetine did not affect the ion selectivity of this K+ channel, as the reversal potential was unaltered. 4 Slowing of both activation and deactivation kinetics of the channel by fluoxetine was observed, including tail current crossover upon repolarization. 5 Hodgkin‐Huxley type of models and more generalized Markov chain models were used to fit the kinetics of the data. Based upon a Markov kinetic scheme, our data can be interpreted to mean that blockade of fluoxetine consists of two components: a voltage‐independent occurring in the last closed, but available state of the channel, and a voltage‐dependent occurring in the open state. 6 This study describes the first biophysical working model for the mechanism of action of fluoxetine on a neuronal, voltage‐dependent K+ channel, RCK1. Although this channel is not very potently blocked by fluoxetine when expressed in oocytes, this study may help us to understand some of the clinical symptoms seen with elevated serum concentrations of this SSRI.

Norpropoxyphene-induced cardiotoxicity is associated with changes in ion-selectivity and gating of HERG currents

OBJECTIVE Norpropoxyphene (NP) is a major metabolite of propoxyphene (P), a relatively weak mu-opioid receptor agonist. Toxic blood concentrations ranging from 3 to 180 mumol/l have been reported and the accumulation of NP in cardiac tissue leads to naloxone-insensitive cardiotoxicity. Since several lines of evidence suggest that not only block of INa but also IK block may contribute to the non-opioid cardiotoxic effects of P and NP, we investigated the effects of P and NP on HERG channels. HERG presumably encodes IKr, the rapidly-activating delayed rectifier K+ current, which is known to have an important role in initiating repolarization of action potentials in cardiac myocytes. METHODS Using the 2-microelectrode voltage clamp technique we investigated the interaction of P and NP with HERG channels, expressed in Xenopus oocytes. RESULTS Our experiments show that low drug concentrations (5 mumol/l) facilitate HERG currents, while higher drug concentrations block HERG currents (IC50-values of approx. 40 mumol/l) and dramatically shift the reversal potential to a more positive value because of a 30-fold increased Na(+)-permeability. P and NP also alter gating of HERG channels by slowing down channel activation and accelerating channel deactivation kinetics. The mutant S631C nullifies the effect of P and NP on the channels K(+)-selectivity. CONCLUSION P and NP show a complex and unique drug-channel interaction, which includes altering ion-selectivity and gating. Site-directed mutagenesis suggests that an interaction with S631 contributes to the drug-induced disruption of K(+)-selectivity. No specific role of the minK subunit in the HERG block mechanism could be determined.

The dual modulation of GIRK1/GIRK2 channels by opioid receptor ligands.

It is well known that activation of the cloned kappa-opioid receptor by nanomolar concentrations of U50488H (trans-(+/-)-3, 4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl-benzeneacetamide) , a selective kappa-opioid receptor agonist, leads to the opening of GIRK1 channels. In this study, we demonstrate that the cloned kappa-opioid receptor functionally couples to GIRK1/GIRK2 channels (G-protein-coupled inwardly rectifying K(+) channels), mimicking the probable heteromultimeric state of neuronal GIRK channels. We also show that micromolar concentrations of U50488H reduce GIRK1/GIRK2 current through direct GIRK1/GIRK2 channel block in a voltage-independent manner (IC(50)=70.28+/-3.68 microM). Similarly, it was found that propoxyphene, methadone, and naloxone also can block GIRK1/GIRK2 current. In contrast, elevated concentrations of morphine (up to 1 mM) did not cause channel block. The related inwardly rectifying K(+) channel, IRK1, was not affected by elevated concentrations of these drugs. We conclude that nanomolar concentrations of opioid receptor ligands activate GIRK1/GIRK2 channels through a receptor-mediated pathway, while micromolar concentrations of some opioid receptor ligands inhibit GIRK1/GIRK2 channels by direct channel block.

High-performance liquid-chromatography with electrochemical detection of buprenorphine and its major metabolite in urine

A procedure was developed for the simultaneous determination of buprenorphine and its major metabolite. N-desalkylbuprenorphine, by reversed-phase high-performance liquid chromatography with electrochemical detection. The detection limit is about 100 pg/ml for the major metabolite and 250 pg/ml for buprenorphine.

Microanalysis of Carbon Monoxide in Blood by Head-Space Capillary Gas Chromatography

A gas chromatographic procedure for the determination of carboxyhaemoglobin in blood was improved by using a capillary system in combination with a micro thermal conductivity detector. This system is very sensitive with high resolutions. The analytical time is reduced to approximately two minutes. Using sulphuric acid as the liberating agent of carbon monoxide, a gradual and reproducible release of carbon monoxide was seen. This shortens the time in emergency cases for sample preparation.

Evidences for a satiating effect of defatted jojoba meal

Abstract Supplementation of food with increasing doses (3, 5, and 10%) of defatted jojoba meal induced a pronounced dose-dependent food intake reduction in fasted and non-fasted rats. This effect was more pronounced in non-fasted than in fasted rats, which is typical for satiety agents. Water intake was reduced to the pair-fed level in non-fasted rats with free access to water and receiving food supplemented with 10% defatted jojoba meal. This was also observed in fasted rats deprived of water during the fasting period and receiving food supplemented with 10% defatted jojoba meal. The reduced water intake in defatted jojoba meal treated rats was entirely due to the food intake reduction. These observations are in favour of the hypothesis that defatted jojoba meal induces its food intake reduction by stimulating satiety. Possible mechanisms are discussed.

Development of a Fluoroimmunoassay for the Detection of Buprenorphine in Urine

The development of a fluoroimmunoassay for the detection of buprenorphine in urine samples is described. Fluorescein-norbuprenorphine and pseudobuprenorphine, the dimer of buprenorphine, were synthesized as tracer molecules. The antibodies were prepared by coupling the 2-diazobenzoic acid derivative of buprenorphine with bovine serum albumine, using the carbodiimide method. The assay was mainly used for the routine detection of buprenorphine in urine specimens of persons suspected of Temgesic abuse. The minimum detectable dose of the immunoassay was calculated to be 20 ng/mL.

Effect of lanthanum on voltage-dependent gating of a cloned mammalian neuronal potassium channel.

The effect of the trivalent cation lanthanum (La3+) on voltage-dependent gating of a cloned mammalian neuronal Kv1.1 potassium channel was studied under whole-cell voltage-clamp conditions in oocytes of Xenopus laevis. La3+ (100 microM) was found to decrease the potassium currents at all test potentials and to shift the midpoint of the fraction open channels/membrane voltage curve by approximately +20 mV. The opening and closing time constants of Kv1.1 channels were empirically fitted with a 4th power Hodgkin-Huxley formalism, or with mono- and multi-exponentials. It was found that La3+ slowed down the kinetics of activation, speeded up those of deactivation, and shifted the opening kinetics by approximately + 60 mV. Interestingly, all these parameters of channel gating were not affected equally by La3+. Furthermore, amplitudes of the inward tail currents evoked at potentials more negative than the potassium equilibrium potential (E(K+)) were more strongly inhibited by La3+ than those of the outward tail currents evoked at potentials more positive than E(K+). This suggests voltage-dependent block and binding of La3+ to the Kv1.1 channel protein. We conclude that these actions cannot be explained in terms of surface charge considerations alone. Our results provide evidence for a direct interaction with the potassium channel protein, shedding new light on the mechanism of action of this lanthanide.

Development of a radioimmunoassay for the determination of N-desmethylzopiclone in urine

The development of a specific and sensitive radioimmunoassay for the detection of N-desmethylzopiclone in urine samples is described. The assay will be used to pre-screen for N-desmethylzopiclone as an indication of zopiclone intake in a forensic and emergency context. N-Hemisuccinyldesmethylzopiclone has been prepared as a hapten. The immunization of rabbits with the hapten-bovine serum albumin conjugate resulted in the production of highly specific antibodies, showing no significant cross-reactivities with existing drugs. The four-parameter logistic model was used to process the calibration data into a fitting curve (r2 = 0.9979). Intra- and inter-assay relative standard deviations were < 9.1 and < 19.1%, respectively. The minimum detectable dose of the immunoassay was calculated to be 0.1 ng ml-1 (Pooled-variance t-distribution, p = 0.01, degrees of freedom = 14).

Routine detection of buprenorphine in horse urine: possibilities and limitations of the combined use of radioimmunoassay, liquid chromatography and gas chromatography-mass spectrometry

Abstract A radioimmunoassay developed for the determination of buprenorphine in human urine samples was examined for its applicability to screening post-race equine urine specimens. The detection limit of the immunoassay in equine urine is 0.1 ng ml −1 . Confirmation methods using liquid chromatography with electrochemical detection and gas chromatography—mass spectrometry have detection limits of 0.2 ng ml −1 (unchanged buprenorphine and N -desalkylbuprenorphine) and 0.1 ng ml −1 (after hydrolysis), respectively. The procedures were validated after in-field administration of 300 μg of buprenorphine to a test horse. The procedures were also applied to screening a large number of random post-race equine urine samples.