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S Lazareno

Publications and source records attributed to S Lazareno.

At least 37 records · Page 2Linked to original sources
PubMed1996

The affinity of adenosine for the high- and low-affinity states of the human adenosine A1 receptor.

The affinity of adenosine for the human adenosine A1 receptor expressed on Chinese hamster ovary cell membranes has been measured in the presence and absence of GTP. The competitive effect of endogenous adenosine on the binding properties of adenosine A1 receptors was estimated from differences in the binding of N6-cyclohexyladenosine measured in the absence and presence of adenosine deaminase. From these data, the affinity of adenosine for the high- and low-affinity states of the human adenosine A1 receptor (7 x 10(7) and 1.3 x 10(5) M-1, respectively) was calculated.

Adenosine↗
PubMed1996

The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H-antagonist binding produced by GTP which has been observed in essentially all studies of A1 receptors and has been ascribed previously to precoupling of A1 receptors to G-proteins in the absence of agonists.

Adenosine↗
PubMed1996

Stereoselective recognition of the enantiomers of phenglutarimide and of six related compounds by four muscarinic receptor subtypes.

1. We have compared the binding properties of the enantiomers of phenglutarimide (1) and of six related compounds to M1 receptors in NB-OK-1 cells, M2 receptors in rat heart, M3 receptors in rat pancreas and the M4 receptors of rat striatum, with their functional (antimuscarinic) properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2) and guinea-pig ileum (M3) receptors. The binding properties of the enantiomers of three of the compounds were also measured on cloned human m1-m4 receptors expressed by CHO cells, using [3H]-N-methylscopolamine ([3H]-NMS) as radioligand. 2. The high affinity enantiomers behaved as competitive antagonists in binding and pharmacological studies. (S)-phenglutarimide (pKi-M1 = 9.0/9.3) and (R)-thienglutarimide (pKi-M1 = 8.6/9.2) recognized selectively the native M1 > M4 > M3 > M2 receptors in tissues as well as the respective cloned receptors. 3. The pA2 values at the inhibitory heteroreceptors in the rabbit vas deferens, and at the guinea-pig atria and ileum for the seven more potent enantiomers were compatible with the previous classification of these receptors as M1/M4-like, M2 and M3, respectively. 4. Replacement of the phenyl by a thienyl ring or of the diethylamino by a piperidino group in the phenglutarimide molecule did not affect markedly the potencies of the high affinity enantiomer. In contrast, replacement of the phenyl by a cyclohexyl ring decreased 20 fold the active enantiomers potency. Methylation of the piperidine-2,6-dione nitrogen also reduced markedly the eutomers' affinities, more on the M1 than on the other subtypes. 5. The selectivity profiles (recognition of four receptor subtypes) of six of the seven less active enantiomers were different from the corresponding more active enantiomers selectivity profiles, suggesting that the preparations used in this study were pure. However, we cannot not exclude the hypothesis that the batch of (S)-thienglutarimide used in this study was contaminated by less than 0.02% of the eutomer. 6. In contrast with the eutomer binding site, replacement of the phenyl ring by a thienyl or cyclohexyl ring did not affect binding of the low affinity enantiomers to the muscarinic receptor or the [3H]-NMS-receptor complex. The replacement of the diethylamino group by a piperidine ring, and N-methylation of the piperidine-2,6 dione moiety increased slightly these enantiomers' potencies. 7. The muscarinic receptors were extremely stereoselective, and had up to 20000 fold lower affinity for the less active enantiomers. However, the stereochemical requirements of the muscarinic receptor subtypes were different for the enantiomers of compounds 1-7, being most stringent at M1 receptors. 8. The weaker enantiomers behaved as competitive antagonists in pharmacological studies, at least in the concentration-range investigated.

Animals↗
PubMed1995

Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors.

Novel methods of detecting and quantitating cooperative interactions between an agent and both a tritiated (muscarinic) antagonist and the endogenous agonist (acetylcholine), acting at a common (muscarinic) receptor, have been devised. In a semiquantitative protocol, binding data are transformed into affinity ratios (the ratios of the apparent affinity of the ligand in the presence of the agent to the affinity of the ligand alone), which allow estimates to be made of the potency of the agent and its cooperativity with the tritiated antagonist and with the unlabeled ligand. These parameters have been quantitated by detailed binding assays or guanosine-5'-O-(3-[35S]thio)triphosphate functional assays. The kinetic phenomena associated with the allosteric interactions have been exploited in two non-equilibrium binding assays, from which the affinity constants describing the allosteric interactions can be extracted. The different assay methods give quantitatively similar and internally consistent estimates of the parameters describing the cooperative interactions. Using these assays, strychnine has been found to act allosterically at muscarinic receptors. Strychnine has an affinity of approximately 10(5) M-1 at the unliganded m1, m2, and m4 receptors but is 5-10-fold weaker at m3 receptors. It is positively cooperative with N-methylscopolamine at m2 and m4 receptors and exhibits neutral and negative cooperativity with m1 and m3 receptors, respectively. With acetylcholine, it is negatively cooperative but the degree of cooperativity is relatively low (2-7-fold), particularly at m1 and m4 receptors. The methods and equations described should be useful in detecting and quantitating allosteric interactions of agents with the endogenous neurotransmitter at G protein-coupled receptors.

Acetylcholine↗
PubMed1995

Probing of the location of the allosteric site on m1 muscarinic receptors by site-directed mutagenesis.

In an attempt to locate the allosteric site on muscarinic receptors to which gallamine binds, 21 residues in the putative external loops and loop/transmembrane helix interfaces have been mutated to alanine. These residues are conserved in mammalian m1-m5 receptors. All mutant receptors can be expressed in COS-7 cells at high levels and appear to be functional, in that acetylcholine binding is sensitive to GTP. The gallamine binding site does not appear to involve the first, second, and most of the third extracellular loops. Tryptophan-400 and -101 inhibit gallamine binding when mutated to alanine or to phenylalanine and may form part of the allosteric site. Several mutations also affect antagonist binding. Surprisingly, tryptophan-91, a residue conserved in monoamine and peptide receptors, is important for antagonist binding. This residue, present in the middle of the first extracellular loop, may have a structural role in many G protein-coupled receptors. Antagonist binding is also affected by mutations of tryptophan-101 and tyrosine-404 to alanine or phenylalanine. In a helical wheel model, trytophan-101 and tyrosine-404, in conjunction with serine-78, aspartate-105, and tyrosine-408, form a cluster of residues that have been reported to affect antagonist binding when mutated, and they may therefore be part of the antagonist binding site. It is suggested that the allosteric site may be located close to and just extracellular to the antagonist binding site. The binding of methoctramine, an antagonist with allosteric properties, is not substantially affected by mutations at tryptophan-91, -101, and -400 and tyrosine-404, and thus these amino acids are not important for its binding. The binding of himbacine, another antagonist with allosteric properties, is affected by these mutations but in a manner different from that of gallamine or competitive antagonists. It has not been possible to determine whether methoctramine and himbacine bind exclusively to the allosteric site or to both the competitive site and the allosteric site.

Acetylcholine↗
PubMed1993

Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1-m4.

We have studied muscarinic agonist stimulated [35S]GTP gamma S binding and [gamma 32P]GTP hydrolysis (GTPase) in membranes from CHO cells stably transfected with human muscarinic m1-m4 receptors. 'Full' agonists were at least 10-fold more potent at m2 & m4 receptors than at m1 & m3. This pattern was less marked with 'partial' agonists, which had a greater maximal effect at m2 & m4 than at m1 & m3. McN-A343 uniquely was more potent and efficacious at m4 than at m2 receptors. Antagonist affinity constants were estimated by fitting the data from inhibition curves directly to the Schild model. Antagonist affinity estimates were very similar to those measured earlier in binding studies using animal tissues, and confirmed a small degree of m4 selectivity for tropicamide and secoverine. The receptor subtypes activated more than one G-protein subtype; m2 & m4 receptors activated only pertussis (PTX) sensitive G-proteins, while m1 & m3 coupled to both PTX sensitive and insensitive G-proteins. Acetylcholine (ACh) was more potent in stimulating guanine nucleotide exchange in PTX-treated m1 cells than in controls.

Animals↗
PubMed1993

Estimation of antagonist Kb from inhibition curves in functional experiments: alternatives to the Cheng-Prusoff equation.

Recent Principles articles have considered procedures for the functional estimation of an antagonist's dissociation constant from the results of an antagonist inhibition curve in the presence of a fixed concentration of agonist. Leff and Dougall (TiPS 14, 110-112) have derived an equation, analogous to the (generally invalid) Cheng-Prusoff equation, which requires that the agonist concentration--effect curves have the form of a logistic function. In the final article of the series, Sebastian Lazareno and Nigel Birdsall consider this equation in the context of two related methods for analysing functional inhibition curves-a null method that makes no assumptions about the shapes of curves, and a curve-fitting method that provides an estimate of the antagonist Schild slope.

Drug Antagonism↗
PubMed1993

Estimation of competitive antagonist affinity from functional inhibition curves using the Gaddum, Schild and Cheng-Prusoff equations.

1. The estimation of antagonist affinity from functional experiments in which the effect of a fixed agonist concentration is reduced by a range of antagonist concentrations ('functional inhibition curves') has been considered from both a theoretical and experimental viewpoint. 2. Theoretical predictions are compared with results obtained from the stimulation of [35S]-GTP gamma S binding by acetylcholine to membranes of Chinese hamster ovary (CHO) cells stably transfected with human m1-m4 muscarinic receptors, and inhibition of the stimulated binding by pirenzepine and AQ-RA 741. 3. The usual procedure of applying the Cheng-Prusoff correction is shown to be theoretically invalid, and predictions are made of the size and distribution of errors associated with this procedure. 4. A different procedure for estimating antagonist affinity, using the principles of dose-ratio analysis and analogous to use of the Gaddum equation, is found to be accurate and theoretically valid. 5. A novel method of analysis allows accurate estimation of both antagonist affinity and Schild slope, by fitting the combined data from an antagonist inhibition curve and an agonist activation curve directly to a form of the Schild equation (derived by Waud) using non-linear regression analysis. 6. It is shown that the conventional Schild analysis can be enhanced by treating part of the data as a family of inhibition curves and including in the Schild plot dose-ratios estimated from the inhibition curves.

Acetylcholine↗
PubMed1993

Pharmacological characterization of acetylcholine-stimulated [35S]-GTP gamma S binding mediated by human muscarinic m1-m4 receptors: antagonist studies.

1. We have used dose-ratio analysis to estimate functionally the affinity constants (pKb) and Schild slope factors of a range of selective or atypical antagonists at human muscarinic m1-m4 receptors. 2. The functional response was the stimulation by acetylcholine of [35S]-GTP gamma S binding to membranes from Chinese hamster ovary (CHO) cells stably expressing individual receptor subtypes. 3. A novel experimental design and analysis was used which allowed the estimation of affinity and Schild slope factor from a single antagonist inhibition curve, and the results were compared with other methods of analysis, both theoretically valid and invalid. 4. In general, the affinity estimates were very similar to previously reported values obtained in binding studies with animal tissues and cloned human receptors and the Schild slope factors were close to unity. 5. These results demonstrate the validity of the assay and provide no evidence for species differences in antagonist affinity for muscarinic receptor subtypes. 6. The results confirm both the utility of himbacine in distinguishing between m1 and m4 receptors and a previously reported modest m4-selectivity for tropicamide and secoverine. 7. The cholinesterase inhibitor, tacrine (THA), had a potency profile similar to that of gallamine but with less selectivity. Its affinity could not be determined since it had Schild slope factors of about 2 at all subtypes. 8. o-Methoxy-sila-hexocyclium had only a modest selectivity for the m1 subtype.

Acetylcholine↗
PubMed1990

Characterization of muscarinic M4 binding sites in rabbit lung, chicken heart, and NG108-15 cells.

We have carried out an extensive pharmacological characterization of muscarinic binding sites in rabbit lung and chicken heart in parallel with M1, M2, and M3 sites, [3H]Pirenzepine, a selective antagonist at M1 receptors, bound saturably and reversibly to membranes from chicken heart and rabbit lung. These binding sites were not M1 receptors, however, because the cardioselective antagonist himbacine had 10-fold higher affinity at these sites than at [3H]pirenzepine sites in rat and rabbit cortex (true M1 sites). We measured the inhibitory potency of 28 antagonists at [3H]N-methylscopolamine-labeled sites in chicken heart, rabbit lung, rat heart (M2 sites), and rat submandibular gland (M3 sites) and at M1 sites in rat cortex. The sites in rabbit lung were different from M1, M2, and M3 sites, because they had moderate to high affinity for M1-selective compounds (pirenzepine and telenzepine), M2-selective compounds (himbacine and methoctramine), and M3-selective compounds (hexahydrosiladifenidol and 4-diphenylacetoxy-N-methylpiperidine methiodide). The sites in chicken heart resembled most those in rabbit lung, with similar high affinity for secoverine, but they were not the same because tropicamide, diphenylacetoxybutynyl dimethylamine, and [3H]-N-methylscopolamine were more potent in rabbit lung. In a further series of experiments, we compared the affinity of six of the most discriminating antagonists in membranes from rabbit lung and NG108-15 cells, a neuroblastoma-glioma cell line reported to express the muscarinic m4 receptor gene. The antagonists had very similar affinities in the two tissues, the largest discrepancy being that pirenzepine was twice as potent in rabbit lung as in NG108-15 cells. Northern blots using probes designed to discriminate between five species of muscarinic receptor RNA detected only m4 mRNA in rabbit lung. We conclude that rabbit lung contains a muscarinic M4 binding site with a quite distinctive pharmacology and that chicken heart contains a receptor with similarities to the M4 sites. This is the first report to characterize native M4 binding sites in a nonneuronal mammalian tissue.

Animals↗
PubMed1989

Functional and binding studies with muscarinic M2-subtype selective antagonists.

1. The potency of a series of selective muscarinic antagonists has been measured on two functional isolated tissue preparations (rat ileum and atria) and these compared with their potency on a range of binding preparations in order to determine whether the subtypes of M2 receptor measured functionally are the same as those measured in binding studies. 2. On the functional preparations pirenzepine, hexahydrosiladiphenidol (HSD) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) were more potent on the ileum than on the atrium (3 fold, 29 fold and 5 fold respectively), whereas himbacine, AF-DX 116 and methoctramine showed the opposite selectivity (5 fold, 3 fold and 56 fold respectively). Atropine had a similar potency on the ileum and atrium. 3. [3H]-N-methyl scopolamine was used to study M2 binding sites on membranes from rat heart and rat submandibular gland. Each preparation appeared to contain a homogeneous binding site population. The potencies of the five M2 selective antagonists (and pirenzepine) in binding studies to heart membranes were very similar to those observed in functional studies of rat atria (correlation coefficient = 0.98). Similarly the binding to submandibular gland membranes was very similar to that observed in functional studies on rat ileum (correlation coefficient = 0.97). 4. [3H]-pirenzepine was used to examine the binding of these antagonists to M1 binding sites on membranes from rat cerebral cortex. The affinities of 4-DAMP, HSD, AF-DX116 and himbacine at M1 sites were similar to their affinities on the gland. Only pirenzepine and methoctramine had higher affinity on M1 sites than on the gland. 5. Himbacine had a 20 fold lower affinity at M1 binding sites than at heart sites, and it should therefore be an important tool in identifying M1 sites. 6. Inhibition of [3H]-N-methyl scopolamine binding to rat ileum and rat brainstem by M2-selective antagonists was best described by a two-site model. In both cases the major population of sites (70-90%) appeared to be similar to sites found on the heart (correlation coefficients = 0.95 and 0.97). The other site appeared to be similar to that on the submandibular gland (correlation coefficients = 0.96 and 1.00). 7. The correlations observed in these studies in which a range of selective muscarinic antagonists was used lend weight to previous studies indicating the presence of three functionally important muscarinic receptor subtypes, typified by the binding sites studied in the cerebral cortex, submandibular gland and heart. 8. We propose that the sub-classification of the M2 muscarinic receptor into M2 and M3 subtypes on the basis of ligand binding studies should be extended to cover functionally-defined receptors as well.

Animals↗
PubMed1985

Differential effects of selective and non-selective neuroleptics on intracellular and extracellular cyclic AMP accumulation in rat striatal slices.

Cyclic AMP was measured in both striatal slices and in the incubation medium after exposure to dopamine and dopamine antagonist. Dopamine increased cyclic AMP in both tissue and medium. The effect of dopamine was enhanced by sulpiride and domperidone, and to a lesser extent by haloperidol, but alpha-fluphenthixol had only an inhibitory effect. The enhancement by sulpiride was stereoselective and totally suppressed by the D1 antagonist SCH 23390. Cyclic AMP in the medium provided the more sensitive measure of drug effect and increased linearly for up to 20 min., whereas the nucleotide in tissue remained stable or declined after 10 min. It is concluded that: the increase in dopamine-stimulated cyclic AMP efflux caused by D2 antagonists reflects increased intracellular cyclic AMP accumulation rather than an effect on the efflux mechanism; dopamine enhances cyclic AMP accumulation via a D1 receptor, and simultaneously inhibits it through a D2 receptor; and changes in D1 receptor-stimulated cyclic AMP formation in striatum may not be related to the clinical actions of neuroleptics. It remains possible that D2 receptor-mediated inhibition of cyclic AMP accumulation stimulated by a different agonist system may underlie some of the therapeutic actions of dopamine agonists and antagonists.

Animals↗
PubMed1985

Pirenzepine indicates heterogeneity of muscarinic receptors linked to cerebral inositol phospholipid metabolism.

Muscarinic receptor-mediated inositol phospholipid metabolism was examined in rat cerebral cortex and medulla-pons slices prelabelled with 3H-inositol. Carbachol stimulated accumulation of 3H-inositol phosphate in both regions with identical EC50 values though the maximal stimulation was considerably greater in cortex. The carbachol response was potently suppressed by atropine with very similar affinities in both regions. However, the M1 antagonist pirenzepine displayed a 10-fold greater affinity for the cortical carbachol response. The data supports the concept of heterogeneity of cerebral muscarinic receptors and suggests that both M1 and M2 sites may be linked to inositol phospholipid metabolism in brain.

Animals↗
PubMed1983

Effects of GTP on 3H-domperidone binding and its displacement by dopamine in rat striatal homogenates.

The potency of dopamine in displacing 3H-domperidone from rat striatal membranes was reduced by GTP and enhanced by Mg++. GTP also increased specific binding, and this effect was larger in the presence of Mg++, which itself inhibited binding. In differently washed and incubated preparations the magnitude of both GTP and Mg++ effects correlated well with the endogenous dopamine content of the membranes. Na+ reduced the potency of dopamine, the GTP effect and specific binding. Saturation experiments revealed that these effects of ions and GTP reflected changes in both the apparent Bmax and KD of 3H-domperidone. It was possible to eliminate the GTP effect on specific binding by extensive washing and incubation, but this treatment may also alter other receptor binding characteristics. It remains unclear whether the enhancing effect of GTP reflects contamination of endogenous dopamine or a genuine property of the D2 receptor.

Animals↗