Biomedical subjects

C Ulens

Publications and source records attributed to C Ulens.

9 recordsLinked to original sources

PubMed2001

Morphine-6beta-glucuronide and morphine-3-glucuronide, opioid receptor agonists with different potencies.

Using heterologous expression in Xenopus laevis oocytes, we compared the potencies of morphine, morphine-6beta-glucuronide (M6G), and morphine-3-glucuronide (M3G) for cloned human mu- (hMOR), kappa- (hKOR), and delta-opioid receptors (hDOR). Each receptor subtype was individually co-expressed with heteromultimeric G-protein coupled inwardly rectifying K(+) (GIRK) channels, consisting of GIRK1 and GIRK2 subunits, and RGS4, a regulator of G-protein signaling. The two-microelectrode voltage clamp technique was used to measure the opioid receptor-activated GIRK1/GIRK2 channel responses. Compared with morphine, M6G had higher potency at the hMOR, lower potency at the hKOR, and similar potency at the hDOR, while M3G showed a 1000-fold lower and non-selective potency via opioid receptors. In contrast to naloxone, M3G did not antagonize the effects of morphine at the hMOR. We also investigated whether Trp318 and His319 provide the molecular basis for mu/delta selectivity and mu/kappa selectivity of morphinan alkaloids by mutating these residues to their corresponding residues in kappa- and delta-opioid receptors. A single-point mutation (W318L) on hMOR completely conferred delta-like potency for morphine and M6G on the mutant mu-receptor. Double mutation at Trp318 and His319 positions (Trp318Y/His319Y) only partially conferred kappa-like potency for morphine and M6G; the decrease in potency for M6G was significantly larger than for morphine. The results of our study show that both M6G and M3G are opioid receptor agonists with different potencies and that the potency of morphinan receptor ligands can be changed by selective mutations of hMOR at the Trp318 and His319 positions.

Analgesics, Opioid↗

PubMed2001

New polypeptide components purified from mamba venom.

New polypeptide components have been isolated from Dendroaspis angusticeps venom using chromatography. Two polypeptides containing 59 and 57 amino acids, called 'DaE1' and 'DaE2' respectively, have been purified to homogeneity and fully sequenced. Spectrometric analysis yielded masses of 6631.5 and 6389.0 Da, respectively. The polypeptides share 98 and 95% identity, respectively, with trypsin inhibitor E (DpE) of Dendroaspis polylepis polylepis. 'DaE' polypeptides inhibit Kv1.1 channels with an IC(50) value in the range of 300 nM. They can be considered as new dendrotoxins, albeit with fairly low affinity as compared to alpha-DTX. 'DaE' polypeptides do not affect Kir2.1 channels.

Animals↗

PubMed2001

Gi- and Gs-coupled receptors up-regulate the cAMP cascade to modulate HCN2, but not HCN1 pacemaker channels.

A hallmark of native pacemaker channels is their regulation by neurotransmitters and hormones acting through the second messenger cAMP. In this study, we investigated the modulation of two cloned pacemaker channels, HCN1 and HCN2, by activation of coexpressed inhibitory G protein (Gi)-coupled (p-opioid) or stimulatory G protein (Gs)-coupled [serotonin 5-HT4(a)] receptors in Xenopus oocytes. Both receptors enhanced HCN2, but not HCN1 currents. Receptor activation increased HCN2 current amplitude, increased the activation rate sixfold and decreased the deactivation rate two-fold. In addition, the fully-activated current for HCN2 increased due to a receptor-induced increase of the maximal conductance. These effects were inhibited by 9-(tetrahydro-2'-furyl)adenine (SQ22536), were independent of protein kinases A and C and could be explained by a cAMP-induced shift of the voltage dependence of activation by 15 mV to more positive potentials. The pathway through which these effects occurred involved Gbetagamma-activation of adenylyl cyclase and, in the case of the p-opioid receptor, required co-expression of Galphas. The effect of the 5-HT4(a)-receptor, in part caused by its constitutive activity, occurred directly through Galphas-activation. This suggests that 5-HT4(a) receptors may contribute to functional heterogeneity of pacemaker currents (Ih) in those neurons in which 5-HT4(a)R and HCN2 coexist.

Adenine↗

PubMed2000

Functional heteromerization of HCN1 and HCN2 pacemaker channels.

An important step toward understanding the molecular basis of the functional diversity of pacemaker currents in spontaneously active cells has been the identification of a gene family encoding hyperpolarization-activated cyclic nucleotide-sensitive cation nonselective (HCN) channels. Three of the four gene products that have been expressed so far give rise to pacemaker channels with distinct activation kinetics and are differentially distributed among the brain, with considerable overlap between some isoforms. This raises the possibility that HCN channels may coassemble to form heteromeric channels in some areas, similar to other K(+) channels. In this study, we have provided evidence for functional heteromerization of HCN1 and HCN2 channels using a concatenated cDNA construct encoding two connected subunits. We have observed that heteromeric channels activate several-fold faster than HCN2 and only a little slower than HCN1. Furthermore, the voltage dependence of activation is more similar to HCN2, whereas the cAMP sensitivity is intermediate between HCN1 and HCN2. This phenotype shows marked similarity to the current arising from coexpressed HCN1 and HCN2 subunits in oocytes and the native pacemaker current in CA1 pyramidal neurons. We suggest that heteromerization may increase the functional diversity beyond the levels expected from the number of HCN channel genes and their differential distribution.

Animals↗

PubMed2000

Changes in GIRK1/GIRK2 deactivation kinetics and basal activity in the presence and absence of RGS4.

The effect of RGS4, a GTPase-activating protein, on the deactivation kinetics and basal activity of GIRK1/GIRK2 channels activated by the human kappa-opioid receptor (hKOR) was investigated. Co-expression in Xenopus oocytes of RGS4 reduces the basal GIRK1/GIRK2 current and strongly increases the percentage agonist-evoked K+ conductance. RGS4 reconstitutes the native gating kinetics by accelerating GIRK1/GIRK2 channel deactivation, a phenomenon also seen after activation with other 7 TM receptors (e.g. muscarine type). In the absence of RGS4, the GIRK1/GIRK2 conductance was increased by approx. 50% after hKOR stimulation with the kappa-selective opioid receptor ligand, U69593; however more importantly, at the end of the washout period it was dramatically reduced to about 60% of the basal conductance as measured before receptor stimulation. Furthermore, we found that repeated receptor stimulation causes an increase of the agonist-gated deactivation kinetics, without affecting the maximal and minimal conductance levels of GIRK1/GIRK2 channels during and after agonist application. Unlike in the absence of RGS4, coexpression with RGS4 completely abolished the reduction of basal conductance after agonist washout and the deactivation kinetics remained unaffected upon repeated agonist application. The results presented here clearly indicate that previous stimulation by agonists activating G protein-coupled receptors may have long-lasting, strong consequences on the following responses. Therefore, our study provides evidence for a novel modulation of deactivation kinetics of GIRK1/GIRK2 currents in the absence of RGS4.

Animals↗

PubMed2000

Redox state dependency of HERGS631C channel pharmacology: relation to C-type inactivation.

The S631C mutation in human ether-à-go-go-related gene (HERG) channels has previously been reported to disrupt C-type inactivation and ion-selectivity when Cys-631 is in the oxidized state. In this study, we report the relation between pharmacology and C-type inactivation for HERGS631C channels. We demonstrate that HERGS631C in its reduced state is fully blocked by 1 microM astemizole, terfenadine and dofetilide, similar to wild-type HERG channels. In contrast, oxidized HERGS631C is insensitive for these blockers. Our results suggest that an interaction with HERG channels in the inactivated state might be a common mechanism to a variety of drugs known to block HERG channels with high affinity.

Animals↗

PubMed2000

Interaction of p-fluorofentanyl on cloned human opioid receptors and exploration of the role of Trp-318 and His-319 in mu-opioid receptor selectivity.

In this study, we investigated the interactions of p-fluorofentanyl, an opioid designer drug, fentanyl, sufentanyl, and morphine on cloned human mu-, kappa-, and delta-opioid receptors coexpressed with heteromultimeric G protein-coupled inwardly rectifying K(+) channels (GIRK1/GIRK2) and a regulator of G protein signaling (RGS4) in Xenopus oocytes. We demonstrate that p-fluorofentanyl more potently activates GIRK1/GIRK2 channels through opioid receptors than fentanyl and that the p-fluoro substitution also changes the potency profile from mu > kappa > delta (fentanyl) to mu > delta > or = kappa (p-fluorofentanyl). A comparison of ligand efficacy revealed that morphine, fentanyl, and its analogs less efficiently activate GIRK1/GIRK2 channels through human mu-opioid receptor than [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin. Using site-directed mutagenesis, we investigated whether mutating residues Trp-318 and His-319 to their corresponding residues in kappa- and delta-opioid receptors provides the molecular basis for mu/delta selectivity and mu/kappa selectivity. Changes in EC(50) values for the W318L and W318Y/H319Y mu-opioid receptors show a partial contribution of these residues to the decreased GIRK1/GIRK2 channel activation by fentanyl analogs through kappa- and delta-opioid receptors. The most pronounced effect was observed for p-fluorofentanyl, suggesting that an interaction between the 4-fluorophenylpropanamide moiety of the drug and residues Trp-318 and His-319 is important for the resulting enhanced GIRK1/GIRK2 channel activation through the mu-opioid receptor. Finally, we demonstrate that mutation of W318L confers delta-like potency for morphine on the mutant mu-opioid receptor.

Animals↗

PubMed1999

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.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1999

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 channel's 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.

Animals↗