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A P Ijzerman

Publications and source records attributed to A P Ijzerman.

At least 19 recordsLinked to original sources
PubMed2001

Extracellular adenosine-induced apoptosis in mouse neuroblastoma cells: studies on involvement of adenosine receptors and adenosine uptake.

The induction of apoptosis by adenosine was studied in the mouse neuroblastoma cell line N1E-115. Apoptosis was characterized by fluorescence and electron microscopy, fluorescence-activated cell sorter (FACS) analysis, and caspase activity assays. A sixteen-hour exposure to 100 microM of adenosine led to chromatin condensation and caspase activation. However, selective agonists for all four adenosine receptors were ineffective. Caspase activation could be blocked partially by an inhibitor of the nucleoside transporter, dipyridamole, and completely by uridine, a competing substrate for adenosine transport. 2'-Deoxycoformycin, an inhibitor of adenosine deaminase, enhanced caspase activation by adenosine but had no effect by itself. Caspase activation could be blocked by 5'-amino-5'-deoxyadenosine, which inhibits the phosphorylation of adenosine by adenosine kinase. These results indicate that adenosine receptors are not involved in adenosine-induced apoptosis in N1E-115 cells, but that uptake of adenosine and its subsequent phosphorylation is required.

Adenosine↗
PubMed2001

Allosteric modulation of A(3) adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives.

Allosteric modulators of A(1) and A(2A) adenosine receptors have been described; however, for the A(3) adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A(3) adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [(125)I]N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [(125)I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [(3)H]PSB-11 from the A(3) adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A(1) receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [(125)I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [(3)H]N(6)-[(R)-phenylisopropyl]adenosine from the A(1) adenosine receptor or [(3)H]CGS21680 from the A(2A) adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.

Adenosine↗
PubMed2000

2-Nitro analogues of adenosine and 1-deazaadenosine: synthesis and binding studies at the adenosine A1, A2A and A3 receptor subtypes.

The influence of nitro substituents on the properties of adenosine and 1-deazaadenosine was studied. Combination of a nitro group at the 2-position with several N6 substituents such as cyclopentyl and m-iodobenzyl gave a series of analogues with good adenosine receptor affinity, showing directable selectivity for the A1, A2A and A3 adenosine receptor subtypes.

Adenosine↗
PubMed2000

Site-directed mutagenesis studies of human A(2A) adenosine receptors: involvement of glu(13) and his(278) in ligand binding and sodium modulation.

To provide insights into interactions between ligands and A(2A) adenosine receptors, site-directed mutagenesis was used to test the roles of a glutamic acid residue in the first transmembrane domain (Glu13) and a histidine residue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, were mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturation experiments revealed that [(3)H]ZM241385 (4-2-[7-amino-2-(2-furyl)-1,2, 4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol) bound wild-type and mutant receptors in membranes from COS-7 cells expressing human A(2A) adenosine receptors with high affinity and low non-specific binding. It was found from the competition experiments that the affinity of the A(2A) adenosine receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3-fold) except in the case of theophylline, whose affinity was decreased approximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [(3)H]ZM241385 were shifted to the right in both wild-type and mutant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mutant receptors. Sodium ions also decreased [(3)H]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former than the latter. The results suggest that the two closely linked residues Glu13 and His278 in A(2A) adenosine receptor are most important for agonist recognition and are partly responsible for the allosteric regulation by sodium ions.

Animals↗
PubMed2000

Allosteric modulation of A(2A) adenosine receptors by amiloride analogues and sodium ions.

Allosteric regulation of rat A(2A) adenosine receptors by amiloride, amiloride analogues, and sodium ions was studied by investigating their ability to influence the dissociation of [(3)H]4-2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a][1,3, 5]triazin-5-yl-amino]ethylphenol ([(3)H]ZM241385) from receptors in rat striatal membranes. Both amiloride and its analogues accelerated the dissociation, the analogues being more potent than amiloride itself. In contrast, sodium ions decreased the rate of [(3)H]ZM241385 dissociation in a concentration-dependent manner, and this rate was not influenced by guanosine triphosphate, N-ethylmaleimide, suramin, or the selective A(2A) adenosine receptor antagonist, 5-amino-2-(2-furyl)-7(2-phenylethyl)pyrazolo[4,3-e]-1,2, 4-triazolo[1,5-c]pyrimidine (SCH58261). The effect of competition between the amiloride analogue 5-(N,N-hexamethylene)amiloride (HMA) and sodium ions on [(3)H]ZM241385 dissociation was also explored. The addition of sodium ions resulted in a concentration-dependent rightward shift of the HMA response curve. The slopes of the HMA concentration-response curves in the presence and absence of sodium ions were not significantly different, which suggests that sodium ions and amiloride analogues act at a common allosteric site on the A(2A) adenosine receptor. There was a lack of correlation between the displacement of ligand binding and the allosteric potencies of the amiloride analogues.

Allosteric Regulation↗
PubMed2000

Why are A(2B) receptors low-affinity adenosine receptors? Mutation of Asn273 to Tyr increases affinity of human A(2B) receptor for 2-(1-Hexynyl)adenosine.

Adenosine A(2B) receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despite numerous synthetic efforts, 5'-N-ethylcarboxamidoadenosine (NECA) still is the reference agonist, albeit nonselective for this receptor. In our search for higher affinity agonists, we developed decision schemes to select amino acids for mutation to the corresponding residues in the most homologous, higher affinity, human A(2A) receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second scheme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the affinity for NECA, (R)-N(6)-(phenylisopropyl)adenosine (R-PIA), and 2Cl Ado. The affinity of the N273Y mutant for NECA and R-PIA and for the antagonists xanthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. However, this mutant had a slightly increased affinity for a 2-substituted adenosine derivative, CGS21680. This prompted us to investigate other 2-substituted adenosines, with selectivity and high affinity for A(2A) receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl)adenosine had submicromolar affinity for the N273Y receptor, 0.18 +/- 0.10 microM, with a 61-fold affinity gain over the wt receptor. In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 +/- 0.5 microM for the wt receptor. The high affinity of (S)-PHP adenosine for the wt receptor suggests that further modifications at the 2-position may yield agonists with even higher affinity for A(2B) receptors.

Adenosine↗
PubMed1999

Study of interaction between agonists and asn293 in helix VI of human beta(2)-adrenergic receptor.

Previously, we demonstrated the involvement of Asn293 in helix VI of the human beta(2)-adrenergic receptor in stereoselective agonist recognition and activation. In the present study, we have further explored the role of this residue by synthesizing derivatives of isoproterenol and clenbuterol, two beta-adrenergic receptor agonists. We analyzed their efficacy and affinity on the wild-type and a mutant receptor (Asn293Leu). Each compound had similar efficacy (tau values) on both the wild-type and mutant receptor, although tau values varied considerably among the eight compounds studied. It appeared that one derivative of isoproterenol, but not of clenbuterol, showed a gain in affinity from the wild type to the mutant receptor. This derivative had a methyl substituent instead of the usual beta-OH group in the aliphatic side chain of isoproterenol, compatible with the more lipophilic nature of the leucine side chain. Such a "gain of function" approach through a combination of synthetic chemistry with molecular biology, may be useful to enhance our insight into the precise atomic events that govern ligand-receptor interactions.

Adrenergic beta-Agonists↗
PubMed1999

Isolation of opioid-active compounds from Tabernaemontana pachysiphon leaves.

A procedure for prefractionation of crude plant extracts by centrifugal partition chromatography (CPC) has been developed to enable rapid identification of known-positive compounds or false-positive compounds and to increase the chance of identifying minor unknown-active compounds. The study explored the use of CPC as a tool in the prefractionation step before investigation of bioactivity. Fractions obtained by CPC from an ethanolic extract of Tabernaemontana pachysiphon Stapf (Apocynaceae) were screened by means of an opiate-receptor-binding assay and an adenosine A1-receptor-binding assay. Fractions containing fatty acids, which had false-positive effects on the assay, were identified, as were unknown-positive fractions from which two opioid-active compounds, tubotaiwine and apparicine, were subsequently isolated. The affinities (Ki) of tubotaiwine and apparicine at the opiate receptor were 1.65 +/- 0.81 and 2.65 +/- 1.56 micromol, respectively. Both alkaloids had analgesic activity in the abdominal constriction test in mice. CPC prefractionation led to the rapid isolation of two opioid-active compounds, tubotaiwine and apparicine, from the unknown-positive fraction; false-positive fractions were rapidly identified. Both tubotaiwine and apparicine had affinity for adenosine receptors in the micromolar range and also had in-vivo analgesic activity in mice.

Alkaloids↗
PubMed1999

Mechanism-based pharmacokinetic-pharmacodynamic modeling of antilipolytic effects of adenosine A(1) receptor agonists in rats: prediction of tissue-dependent efficacy in vivo.

In this study, we analyzed the antilipolytic effects of six N(6)-cyclopentyladenosine analogs in rats and developed a mechanistic pharmacokinetic-pharmacodynamic model to quantify and predict the tissue-selective action of adenosine A(1) receptor agonists in vivo. Freely moving rats received an i.v. infusion of vehicle or compound over 15 min. Arterial blood samples were taken at regular time intervals for the determination of concentrations of drugs using HPLC analysis and of nonesterified fatty acids (NEFAs). All N(6)-cyclopentyladenosine analogs that were investigated produced a significant decrease in the NEFA plasma concentration after i.v. infusion. The pharmacokinetic behavior of each ligand was described by a standard two-compartment model. The pharmacokinetic parameter estimates then were used to simultaneously fit the individual (n = 6-8) time-NEFA concentration profiles for each agonist to a physiological indirect response model in combination with the Hill equation to obtain estimates of the NEFA elimination rate constant (k(e)) and upper asymptote (fractional inhibition), midpoint location, and midpoint slope parameter (alpha, pEC(50), and n(H), respectively) of the concentration-effect relationship. Subsequently, the data were analyzed with the operational model of agonism to obtain estimates of in vivo affinity and efficacy. It was estimated that the in vivo density and/or coupling of adenosine A(1) receptors mediating antilipolytic effects is approximately 38 times higher compared with the receptors mediating bradycardia. The model predicts that it is possible to design ligands that produce significant inhibition of lipolysis and are completely devoid of cardiovascular effects in vivo.

Adenosine↗
PubMed1998

A novel class of adenosine A3 receptor ligands. 2. Structure affinity profile of a series of isoquinoline and quinazoline compounds.

1-Substituted 3-(2-pyridinyl)isoquinolines have been shown to form a novel class of adenosine A3 receptor ligands. In the present study further investigations of this new lead and the structure affinity relationships of this class of compounds are described. First, the influence of an amide group at position 1 of the isoquinoline ring on the adenosine A3 receptor affinity was determined. A carboxamide proved to be a useful spacer between the isoquinoline and a phenyl ring. N-[2-(2-pyridinyl)isoquinolin-4-yl]benzamide (VUF8507, compound 6) had an affinity of 200 nM at the adenosine A3 receptor. Second, we investigated the effects of substitution of the benzamide ring of 6 with a series of mono- and disubstituted N-[3-(2-pyridinyl)isoquinoline]benzamides. The ratio of the tautomers of the benzamides was determined in the solid state and in solution by spectroscopic techniques (IR and NMR). Affinities were determined in radioligand binding assays at rat brain A1 and A2A receptors and at cloned human A3 receptor. The benzamides showed higher adenosine A3 receptor affinity than aliphatic amides. We propose that the adenosine A3 receptor affinity of the different benzamides is related to their presence in either the iminol or amide form. Ligands present in the iminol form showed relatively high adenosine A3 receptor affinity. Finally, we explored the influence of replacement of C4 of the isoquinoline ring by a nitrogen atom. Comparison of isoquinolines with the corresponding quinazolines revealed that both compounds showed similar adenosine A3 receptor affinity. These investigations led to potent and selective human adenosine A3 receptor ligands with affinities in the nanomolar range. The subtype-selective compound 4-methoxy-N-[2-(2-pyridinyl)quinazolin-4-yl]benzamide (VUF8504, 13) with an affinity of 17.0 nM at the human adenosine A3 receptor might become a useful tool in the pharmacological characterization or the investigation of the physiological function of this receptor.

Animals↗
PubMed1998

Role of ceramide 1 in the molecular organization of the stratum corneum lipids.

The main barrier of the skin is formed by the lipids in the apical skin layer, the stratum corneum (SC). In SC mainly ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL) are present. The CER are composed of at least six different fractions. CER 1 has an exceptional molecular structure as it contains a linoleic acid linked to a long-chain omega-hydroxy acid (C > 30). The SC lipids are organized in two lamellar phases with periodicities of approximately 6 and 13 nm, respectively. Recent studies revealed that ceramides isolated from pig SC mixed with cholesterol in confined ratios mimic stratum corneum lipid phase behavior closely (Bouwstra, J.A., et al. 1996. J. Lipid Res. 37: 999-1011). In this paper the role of CER 1 for the SC lipid lamellar organization was studied. For this purpose lipid phase behavior of mixtures of CHOL and total ceramide fraction was compared with that of mixtures of CHOL and a ceramide mixture lacking CER 1. These studies showed that in the absence of CER 1 almost no long periodicity phase was formed over a wide CHOL/CER molar ratio. A model is proposed for the molecular arrangement of the two lamellar phases. This model is based on the dominant role CER 1 plays in the formation of the long periodicity phase, electron density distribution calculations, and observations, such as i) the bimodal distribution of the fatty acid chain lengths of the ceramides, ii) the phase separation between long-chain ceramides and short-chain ceramides in a monolayer approach, and iii) the absence of swelling of the lamellae upon increasing the water content organization in SC. In this molecular model the short periodicity phase is composed of only two high electron density regions indicating the presence of only one bilayer, similar to that often found in phospholipid membranes. The molecular arrangement in the long periodicity phase is very exceptional. This phase most probably consists of two broad and one narrow low electron density regions. The two broad regions are formed by partly interdigitating ceramides with long-chain fatty acids of approximately 24-26 C atoms, while the narrow low-electron density region is formed by fully interdigitating ceramides with a short free fatty acid chain of approximately 16 to 18 C atoms.

Animals↗
PubMed1998

Metabolic and cardiovascular effects of the adenosine A1 receptor agonist N6-(p-sulfophenyl)adenosine in diabetic Zucker rats: influence of the disease on the selectivity of action.

Studies were designed to investigate differences in pharmacokinetics and pharmacodynamics of the adenosine A1 receptor agonist N6-(p-sulfophenyl)adenosine (SPA) between lean and obese Zucker rats. In conscious rats, time courses of the effect on heart rate and parameters of lipid metabolism (fatty acids, glycerol) were monitored in combination with the decline of drug concentrations after i.v. administration of 100 microgram SPA in 15 min. Small differences in pharmacokinetics of SPA were observed between lean and obese rats. Values for clearance and volume of distribution were 1.2 +/- 0.2 ml/min and 88 +/- 10 ml in lean, and 1.6 +/- 0.1 ml/min and 110 +/- 7 ml in obese animals, respectively. Modelling of the concentration-heart rate relationship on the basis of the sigmoidal Emax model revealed no difference in EC50 (99 +/- 12 and 118 +/- 17 ng/ml) or Emax (-191 +/- 16 and -185 +/- 22 bpm) between the lean and obese rats. The metabolic effects of SPA were totally different between lean and obese rats. Potent (EC50 = 18 +/- 3 ng/ml) inhibition of lipolysis was observed in the lean rats. In obese rats, SPA was less potent (EC50 = 109 +/- 36 ng/ml) resulting in short lasting antilipolytic effect. Furthermore, administration of SPA resulted in a significant decrease in insulin concentrations. These findings show that changes in glucose and lipid metabolism may be associated with an altered sensitivity to the antilipolytic actions of adenosine A1 receptor agonists.

Adenosine↗
PubMed1997

Physiological indirect effect modeling of the antilipolytic effects of adenosine A1-receptor agonists.

The relationship between blood concentrations of the adenosine A1-receptor agonist N6-(p-sulfophenyl) adenosine (SPA) and its effect on both plasma nonesterified fatty acid (NEFA) and glycerol release was described on the basis of an integrated pharmacokinetic-pharmacodynamic model. An indirect response model rather than a hypothetical "link" model was used to account for the delayed response. For that purpose an empirical solution to the differential equation describing the physiological indirect response model is presented. The model-estimated rate constant for the output of the glycerol response was compared to the elimination rate constant after exogenous administration of glycerol. In a crossover designed study, chronically cannulated male Wistar rats were subjected to either SPA administration (120 microgram/kg for 15 min) for measurement of the effects on glycerol, or glycerol administration for determination of glycerol pharmacokinetics. Glycerol pharmacokinetics was determined in the presence of a stable level of SPA (171 +/- 6 ng/ml) to suppress endogenous glycerol levels completely. The indirect response model adequately described the relationship between SPA concentrations and plasma glycerol levels. The PD parameter estimates for EC50, EMAX, and Hill factor were 23 +/- 2 ng/ml, 74 +/- 3% (change from baseline), and 3.3 +/- 0.5, respectively. These values were not different from those obtained when analyzing the data on basis of the differential equation directly. Furthermore, the EC50 values for the reduction in glycerol or NEFA levels were identical (23 +/- 2 and 21 +/- 3 ng/ml, respectively) indicating that both PD endpoints reflect the same physiological process. The concentration-time profile after administration of glycerol could be described best on the basis of a biexponential function. The value for kout in the PK/PD model (0.19 +/- 0.03 min-1) corresponded very well to the terminal elimination rate constant determined after i.v. administration of glycerol (0.25 +/- 0.03 min-1). In conclusion, the antilipolytic effects of adenosine A1-receptor agonists can be described by the indirect suppression model. The rate constant describing the delay between concentration and glycerol effect was shown to be a true reflection of the removal of glycerol.

Adenosine↗
PubMed1997

Study of the interaction between aryloxypropanolamines and Asn386 in helix VII of the human 5-hydroxytryptamine1A receptor.

We studied the stereoselective interaction between aryloxypropanolamines and the human 5-hydroxytryptamine1A (5-HT1A) receptor. R- and S-enantiomers of propranolol, penbutolol, and alprenolol were investigated for their ability to bind to human 5-HT1A wild-type and Asn386Val mutant receptors. Asn386 seemed to act as a chiral discriminator. Although both aryloxypropanol enantiomers displayed lower affinity for the mutant receptors, the affinities for the S-enantiomers were more affected. Receptor affinities of other structurally unrelated 5-HT1A ligands were not decreased by the mutation of Asn386 to valine. In addition, a series of analogues of propranolol with structural variation in the oxypropanolamine moiety was synthesized, and affinities for wild-type and Asn386Val mutant 5-HT1A receptors were determined. Both the hydroxyl and the ether oxygen atoms of the oxypropanol moiety seem to be required for binding at wild-type 5-HT1A receptors. The hydroxyl group of propranolol probably directly interacts with Asn386. The ether oxygen atom may be important for steric reasons but can also be involved in a direct interaction with Asn386. These findings are in agreement with the interactions of aryloxypropanolamines with Asn386 in rat 5-HT1A receptors that we previously proposed. The loss of affinity for propranolol by the Asn386Val mutation could be regained by replacement of the hydroxyl group of the ligand by a methoxy group. This modification of the propranolol structure has no effect on the affinity of both enantiomers for the wild-type 5-HT1A receptor, which provides an alternative hypothesis for the interaction of Asn386 with the oxypropanol oxygen atoms. According to this novel hypothesis, the oxypropanol oxygen atoms may both act as hydrogen bond acceptors from the NH2 group of Asn386.

Alprenolol↗
PubMed1997

8-Alkylamino-substituted analogs of N6-cyclopentyladenosine are partial agonists for the cardiovascular adenosine A1 receptors in vivo.

Partial adenosine A1 receptor agonists with reduced intrinsic activity at the cardiovascular system would be promising for therapeutic application (e.g., as antilipolytic agents). In the present study a series of 8-alkylamino [methyl (M)-, ethyl (E)-, propyl (P)-, butyl (B)- and cyclopentyl (CP)-] derivatives of N6-cyclopentyladenosine (CPA) were investigated in conscious normotensive rats. After intravenous administration of the compounds to rats, heart rate (HR) and mean arterial pressure were monitored continuously, and serial arterial blood samples were drawn for determination of the pharmacokinetics. The concentration-heart rate relationships of the compounds were described on the basis of an integrated pharmacokinetic-pharmacodynamic model. The blood concentration-time profiles of the compounds could be described best by a biexponential function. The derivatives of CPA had uniform pharmacokinetic properties. The larger volume of distribution at steady state of the 8-substituted analogs resulted in terminal half-lives (ranging from 17 to 24 min) which were significantly longer than for CPA (7 min). All derivatives of CPA produced less pronounced reductions in HR and MAP than CPA. The relationship between concentration and the reduction in HR was adequately described by the sigmoidal Emax model in individual rats given 8MCPA, 8ECPA and 8PCPA. 8BCPA and 8CPCPA were nearly inactive on heart rate. The in vivo EC50,u values for the reduction in HR (366 nM, 210 nM, 170 nM and 175 nM for 8MCPA, 8ECPA, 8PCPA and 8BCPA, respectively) were in the same order of magnitude as the affinities in receptor binding studies. The order of magnitude of the intrinsic activities (Emax) was CPA > 8MCPA > 8ECPA = 8PCPA > 8BCPA > 8CPCPA, which indicated partial agonism of the compounds in vivo. The in vivo parameter Emax correlated highly (r = 0.97) to the GTP shift observed in radioligand binding experiments.

Adenosine↗