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Dive into the research topics where Philip Wahl is active.

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Featured researches published by Philip Wahl.


The Journal of Physiology | 1997

Control of rat GluR6 glutamate receptor open probability by protein kinase A and calcineurin

Stephen F. Traynelis; Philip Wahl

1 We have used non‐stationary variance analysis to examine the single channel conductance and the probability of channel opening at the peak of the homomeric GluR6 response (Po,peak) to 100–200 ms application (10–90% exchange time, 0–3 ms) of glutamate onto excised membrane patches from transiently transfected human embryonic kidney cells (HEK 293). 2 Our determinations of both Po,peak and single channel conductance of simulated current responses are insensitive to system filtering, response rise time, desensitization rate and measured variation in our drug perfusion speed. Isolation of stochastic current fluctuations using the local mean response waveform minimizes problems associated with modest run‐down of response amplitude during the experiment. 3 The slope conductance calculated from the weighted mean unitary currents for the channels activated in response to glutamate application is 16 pS. Chord conductance between −40 and −80 mV is independent of agonist concentration. Conversion of the codon for glutamine621 to arginine (Q621R) by RNA editing reduces conductance by more than 35‐fold to less than 0.4 pS without changing response time course, desensitization, or Po,peak. 4 P o,peak is high at saturating glutamate concentrations (0.65 ± 0.23; mean ±s.d.) and varies with agonist concentration. The half‐maximally effective glutamate concentration (EC50) determined for Po,peak (0.2 mm; Hill slope = 0.6) is similar to that determined for the macroscopic peak current amplitude (0.5 mm; Hill slope = 1.0) in response to rapid agonist application. 5 Inclusion of the purified catalytic subunit of cAMP‐dependent protein kinase A (PKA) in the patch pipette increases Po,peak to 0.85 ± 0.12 and co‐transfection of cells with a cDNA encoding the catalytic subunit of PKA (Cα‐PKA) increases Po,peak to 0.94 ± 0.09. 6 Inclusion of purified calcineurin plus its coactivators 200 nm Ca2+ and calmodulin in the patch pipette decreases Po,peak to 0.48 ± 0.10. The calcineurin‐stimulated decrease of Po,peak in cells co‐transfected with Cα‐PKA is blocked by 800 nm deltamethrin, a calcineurin inhibitor. Calmodulin, 200 nm Ca2+ and deltamethrin have no effect on Po,peak in the absence of calcineurin. As predicted from its effects on Po,peak, inclusion of calcineurin in the patch pipette accelerates the run‐down of whole cell GluR6 responses in cells co‐transfected with Cα‐PKA. 7 The effects of both calcineurin and PKA on Po,peak for GluR6 receptors in excised patches occur without any detectable changes to response time course, desensitization, or chord conductance. 8 We conclude that the binding of glutamate to homomeric GluR6 receptors is associated with a high probability of channel opening, which is under the control of two signalling systems that are known to be co‐localized at the neuronal membrane: PKA (Po,peak near 1.0) and calcineurin (Po,peak near 0.5).


Diabetologia | 2001

Effect of repaglinide on cloned beta cell, cardiac and smooth muscle types of ATP-sensitive potassium channels.

Michael Dabrowski; Philip Wahl; W. E. Holmes; F M Ashcroft

Aims/hypothesis. The carbamoylbenzoic acid derivative repaglinide is a potent short-acting insulin secretagogue that acts by closing ATP-sensitive potassium (KATP) channels in the plasma membrane of the pancreatic beta cell. In this paper we investigated the specificity of repaglinide for three types of cloned (KATP) channel composed of the inwardly rectifying potassium channel Kir6.2 and either the sulphonylurea receptor SUR1, SUR2A or SUR2B, corresponding to the beta cell, cardiac and either smooth muscle types of KATP channel, respectively. Methods. The action of the drug was studied by whole-cell current recordings of KATP channels expressed either in Xenopus oocytes or mammalian cells (HEK293). We also used inside-out macropatches excised from Xenopus oocytes for detailed analysis of repaglinide action. Results. The drug blocked all three types of KATP channel with similar potency, by interacting with a low-affinity site on the pore-forming subunit of the channel (Kir6.2: half-maximal inhibition 230 μmol/l) and with a high-affinity site on the regulatory subunit, the sulphonylurea receptor (SUR: half-maximal inhibition 2–8 nmol/l). There was no difference in potency between channels containing SUR1, SUR2A or SUR2B. MgADP potentiated the inhibitory effect of repaglinide on Kir6.2/SUR1 and (to a lesser extent) Kir6.2/SUR2B, but not on Kir6.2/SUR2A. Conclusion/interpretation. Repaglinide interacts with a site common to all three types of sulphonylurea receptor leading to inhibition of the KATP channel. The fact that MgADP potentiated this effect in the case of the beta cell, but not cardiac, type of channel could help explain why the drug shows no adverse cardiovascular side-effects in vivo. [Diabetologia (2001) 44: 747–756]


Current Medicinal Chemistry | 2004

Inhibition of Insulin Secretion as a New Drug Target in the Treatment of Metabolic Disorders

J. Bondo Hansen; Per Arkhammar; Thora B. Bodvarsdottir; Philip Wahl

The pattern of insulin release is crucial for regulation of glucose and lipid haemostasis. Deficient insulin release causes hyperglycemia and diabetes, whereas excessive insulin release can give rise to serious metabolic disorders, such as nesidioblastosis (Persistent Hyperinsulinemic Hypoglycemia of Infancy, PHHI) and might also be closely associated with development of type 2 diabetes and obesity. Type 2 diabetes is characterized by fasting hyperinsulinemia, insulin resistance and impaired insulin release, i.e. reduced first phase insulin release and decreased insulin pulse mass. The beta cell function of patients with type 2 diabetes slowly declines and will ultimately result in beta cell failure and increasing degrees of hyperglycemia. Type 2 diabetes, in combination with obesity and cardiovascular disorders, forms the metabolic syndrome. It has been possible to improve beta cell function and viability in preclinical models of type 1 and type 2 diabetes by reducing insulin secretion to induce beta cell rest. Clinical studies have furthermore indicated that inhibitors of insulin release will be of benefit in treatment or prevention of diabetes and obesity. Pancreatic beta cells secrete insulin in response to increased metabolism and by stimulation of different receptors. The energy status of the beta cell controls insulin release via regulation of open probability of the ATP sensitive potassium (K(ATP)) channels to affect membrane potential and the intracellular calcium concentration [Ca(2+)](i). Other membrane bound receptors and ion channels and intracellular targets that modulate [Ca(2+)](i)will affect insulin release. Thus, insulin release is regulated by e.g. somatostatin receptors, GLP-1 receptors, muscarinic receptors, cholecystokinin receptors and adrenergic receptors. Although the relationship between hyperinsulinemia and certain metabolic diseases has been known for decades, only a few inhibitors of insulin release have been characterized in vitro and in vivo. These include the K(ATP) channel openers diazoxide and NN414 and the somatostatin receptor agonist octreotide.


British Journal of Pharmacology | 2005

Kir6.2-dependent high-affinity repaglinide binding to β-cell KATP channels

Ann Maria Kruse Hansen; John Bondo Hansen; Richard D. Carr; Frances M. Ashcroft; Philip Wahl

1 The β‐cell KATP channel is composed of two types of subunit – the inward rectifier K+ channel (Kir6.2) which forms the channel pore, and the sulphonylurea receptor (SUR1), which serves as a regulatory subunit. The N‐terminus of Kir6.2 is involved in transduction of sulphonylurea binding into channel closure, and deletion of the N‐terminus (Kir6.2ΔN14) results in functional uncoupling of the two subunits. In this study, we investigate the interaction of the hypoglycaemic agents repaglinide and glibenclamide with SUR1 and the effect of Kir6.2 on this interaction. We further explore how the binding properties of repaglinide and glibenclamide are affected by functional uncoupling of SUR1 and Kir6.2 in Kir6.2ΔN14/SUR1 channels. All binding experiments are performed on membranes in ATP‐free buffer at 37°C. 2 Repaglinide was found to bind with low affinity (KD=59±16 nM) to SUR1 alone, but with high affinity (increased ∼150‐fold) when SUR1 was co‐expressed with Kir6.2 (KD=0.42±0.03 nM). Glibenclamide, tolbutamide and nateglinide all bound with marginally lower affinity to SUR1 than to Kir6.2/SUR1. 3 Repaglinide bound with low affinity (KD=51±23 nM) to SUR1 co‐expressed with Kir6.2ΔN14. In contrast, the affinity for glibenclamide, tolbutamide and nateglinide was only mildly changed as compared to wild‐type channels. 4 In whole‐cell patch‐clamp experiments inhibition of Kir6.2ΔN14/SUR1 currents by both repaglinide and nateglinde is abolished. 5 The results suggest that Kir6.2 causes a conformational change in SUR1 required for high‐affinity repaglinide binding, or that the high‐affinity repaglinide‐binding site includes contributions from both SUR1 and Kir6.2. Glibenclamide, tolbutamide and nateglinide binding appear to involve only SUR1.


European Journal of Pharmacology | 1996

Different characteristics of AMPA receptor agonists acting at AMPA receptors expressed in Xenopus oocytes

Philip Wahl; Ulf Madsen; Tue G. Banke; Povl Krogsgaard-Larsen; Arne Schousboe

A series of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) analogues were evaluated for activity at homo-oligomeric glutamate1-flop (Glu1-flop) receptors expressed in Xenopus oocytes, using the two-electrode voltage clamp technique. (RS)-2-Amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA) (EC50, 2.4 microM), a homologue of AMPA having a carboxyl group as the terminal acidic functionality, was five times more potent than AMPA (EC50, 12 microM) and 20 times more potent than kainate (EC50, 46 microM). (RS)-2-Amino-3(3-hydroxy-5-trifluoromethyl-4-isoxazolyl)propionic acid (Tri-F-AMPA), in which an electronegative trifluoromethyl group is substituted for the methyl group on the isoxazole ring in the AMPA structure, was three times more potent than AMPA, whereas (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-5-carboxylic acid (5-HPCA), a bicyclic analogue of AMPA with highly restricted conformational flexibility was 10 times less potent than AMPA. The limiting slope of log-log plots of Glu1-flop receptor currents versus low agonist concentrations had a value of 1.7 for ACPA and kainate compared to 1.5 for Tri-F-AMPA and 1.3 for 5-HPCA and AMPA. The amplitude of responses evoked by near saturating concentrations of the agonists varied more than 7-fold. The sequence of efficacy was ACPA = kainate > Tri-F-AMPA > AMPA > 5-HPCA. Moreover, when saturating concentrations of Tri-F-AMPA and kainate were co-applied, the response was significantly greater than when each of the agonists was applied separately. The potency of the antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) (estimated KB, approximately 200 nM), to block currents mediated by Glu1-flop receptors was similar for all of the agonists tested in this study. These results indicate that relatively minor changes in the molecular structure of AMPA are associated with marked effects on potency and efficacy. In particular, it is suggested that the acidity of the terminal group plays a major role in determining the degree of receptor activation in the steady state.


Journal of Neurochemistry | 2008

Identification and Function of Glycine Receptors in Cultured Cerebellar Granule Cells

Philip Wahl; Lisbeth Elster; Arne Schousboe

Abstract: Poly(A)+ mRNA was isolated from cultured mouse cerebellar granule cells and injected into Xenopus oocytes. This led to the expression of receptors that evoked large membrane currents in response to glycine. Current‐responses were also obtained after application of β‐alanine and taurine, but these were very low relative to that of glycine (maximal β‐alanine and taurine responses were 8 and 3% of that of glycine, respectively). The role of glycine receptors on K+‐evoked transmitter release in cultured cerebellar granule cells was also assayed. Release of preloaded d‐[3H]aspartate evoked by 40 mM K+ was dose dependently inhibited by glycine, and the concentration producing half‐maximal inhibition was 50 μM. Taurine, β‐alanine, and the specific GABAA receptor agonist isoguvacine also inhibited K+‐evoked release, and the maximal inhibition was similar for all agonists (˜40%). The EC50 value was 200 μM for taurine, 70 μM for β‐alanine, and 4 μM for isoguvacine. Bicuculline (150 μM) antagonized the inhibitory effect of isoguvacine (150 μM) but not that of glycine (1 mM). In contrast, strychnine (20 μM) antagonized the inhibitory effect of glycine (1 mM) but not that of isoguvacine (150 μM). The pharmacology of the responses to β‐alanine and taurine showed that these agonists activate both glycine and GABAA receptors. The results indicate that cultured cerebellar granule cells translate the gene for the glycine receptor and that activation of glycine receptors produces neuronal inhibition.


FEBS Letters | 1998

Glucagon‐like peptide‐1 receptor expression in Xenopus oocytes stimulates inositol trisphosphate‐dependent intracellular Ca2+ mobilization

Jesper Gromada; Charlotte Anker; Krister Bokvist; Lotte Bjerre Knudsen; Philip Wahl

The signal transduction pathway of the cloned human glucagon‐like peptide‐1 (GLP‐1) receptor was studied in voltage‐clamped Xenopus oocytes. Binding of GLP‐1(7–36)amide was associated with cAMP production, increased [Ca2+]i and activation of Ca2+‐dependent Cl− current. The effect of GLP‐1(7–36)amide reflects intracellular Ca2+ mobilization and was suppressed by injection of the Ca2+ chelator BAPTA and the inositol trisphosphate receptor antagonist heparin. The responses were not mimicked by the adenylate cyclase activator forskolin and unaffected by the protein kinase A (PKA) inhibitor Rp‐cAMPS. We conclude that GLP‐1 receptor expression in Xenopus oocytes evokes inositol trisphosphate‐dependent intracellular Ca2+ mobilization independent of the cAMP/PKA signaling pathway.


Neuropharmacology | 1998

Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors

Philip Wahl; A Frandsen; Ulf Madsen; Arne Schousboe; P. Krogsgaard-Larsen

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propi onic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC50 value of 150 microM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC50 value of 170 microM and a relative efficacy of 0.17 +/- 0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED50 > 300 microM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED50 = 14 +/- 2 microM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 microM) or DNQX (25 microM). A structure-activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.


Diabetes | 2007

The Actions of a Novel Potent Islet β-Cell–Specific ATP-Sensitive K+ Channel Opener Can Be Modulated by Syntaxin-1A Acting on Sulfonylurea Receptor 1

Betty Ng; Youhou Kang; Chadwick L. Elias; Yan He; Huanli Xie; John Bondo Hansen; Philip Wahl; Herbert Y. Gaisano

Islet β-cell–specific ATP-sensitive K+ (KATP) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in β-cells to inhibit KATP channels. As a strategy to elucidate the molecular mechanism of action of these KATP channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence syntaxin-1A–SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-syntaxin-1A dialysis or green fluorescence protein/syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on syntaxin-1A–SUR1 interactions. Dialysis of GST–syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet β-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST–syntaxin-1A and its H3 domain inhibited KATP channels previously activated by NNC55-0462. This action on KATP channels is isoform-specific to syntaxin-1A because syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST–syntaxin-1A inhibition. NNC55-0462, however, did not influence syntaxin-1A–SUR1 binding interaction. Our results demonstrated that syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the KATP channel openers NNC55-0462 and diazoxide on KATP channels. The reduced levels of islet syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of KATP channel openers.


Bioorganic & Medicinal Chemistry Letters | 2001

Phenylcyanoguanidines as inhibitors of glucose-induced insulin secretion from beta cells

Tina Møller Tagmose; John Patrick Mogensen; Pia C Agerholm; Per Arkhammar; Philip Wahl; Anne Worsaae; J. Bondo Hansen

3,5-Disubstituted-phenylcyanoguanidines have been identified as activators of SUR1/Kir6.2 potassium channels and as potent inhibitors of insulin release from pancreatic beta cells in vitro.

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Philippe Lebrun

Université libre de Bruxelles

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Arne Schousboe

University of Copenhagen

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