Biomedical subjects

E Leung

Publications and source records attributed to E Leung.

At least 91 records · Page 5Linked to original sources

PubMed1990

EQUIL: simulation and data analysis of binding reactions with arbitrary chemical models.

We have developed an algorithm for simulation and analysis of arbitrary chemical systems in equilibrium, with emphasis on ligand binding reactions. The program EQUIL can treat reactions involving multiple ligands, multiple binding sites, ternary complex models, allosteric effectors, competitive and noncompetitive binding, conformational changes, cooperativity, and generally any scheme that can be represented as a set of chemical equations. EQUIL is based on a general thermodynamic model of chemical equilibria; it does not involve nonlinear transformation of experimental data, but it does require the user to define the model of interaction between ligands and receptors by writing down the appropriate chemical reactions. EQUIL contains features of particular importance to ligand binding experiments: variable binding capacities, nonspecific binding, and the ability to simultaneously analyze data from different types of experiments. Furthermore, the simulation feature of EQUIL allows the user to investigate the feasibility of experiments that could possibly distinguish between different reaction models. We illustrate the use of this program on personal computers to analyze and simulate simple and complicated interactions between ligands and receptors.

Algorithms↗

PubMed1990

Analysis of agonist-antagonist interactions at A1 adenosine receptors.

Previous work from our laboratory using sucrose gradient centrifugation and the antagonist radioligand [3H]xanthine amine congener led us to propose that A1 adenosine receptors are coupled to a GTP-binding protein (G protein) in the absence of an agonist and that adenosine receptor antagonists bind to free uncoupled receptors with high affinity and coupled receptors with low affinity and cause a destabilization of receptor-G protein complexes [Mol. Pharmacol. 36:412-419 (1989)]. Because agonists form high affinity ternary complexes composed of the agonist, receptor, and G protein, this hypothesis would imply that interactions between adenosine receptor agonists and antagonists, while competitive, would appear to be "noncompetitive" in nature. Interactions between unlabeled and radiolabeled A1 receptor agonist and antagonist ligands have been investigated using bovine cerebral cortical membranes to further probe this point. The availability of both 3H- and 125I-radioligands allowed us to use both single- and dual-isotope experimental designs. Radioligand antagonist-agonist competition curves along with saturation analyses using filtration and centrifugation to isolate bound radioligand suggested that agonists bind to two sites or receptor states with high affinity and to one site with low affinity. Agonist radioligand saturation curves with or without unlabeled antagonist suggested that antagonists do not bind to all states of the receptor with equal affinity. The computer program EQUIL was used to define models capable of simultaneously fitting all parts of complex experiments in which 125I-N6-aminobenzyladenosine saturation isotherms with or without 8-cyclopentyl-1,3-dipropylxanthine ([3H]CPX) and a saturation isotherm of [3H]CPX were performed. The data were not compatible with two-independent site models or with ternary complex models involving one receptor and one G protein. The data were fit by a model involving one receptor and two G proteins and by a model involving two receptors and one G protein. Both models suggest that 1) a high percentage of the receptor(s) is coupled to a G protein in the absence of an agonist and 2) agonists stabilize whereas antagonists destabilize precoupled receptor-G protein complexes. Because of this, competitive interactions between A1 agonists and antagonists appear noncompetitive in nature.

Animals↗

PubMed1989

Isolation of murine fetal thymus cell lines after infection with recombinant retroviruses containing the v-myc and v-Ha-ras oncogenes.

The mechanisms that regulate thymocyte proliferation and differentiation within the thymus are still poorly understood. As a novel approach to analyzing these problems, we have used a retroviral vector to insert oncogenes into fetal thymic cells, and construct a library of thymic cell lines. Infection with a double promoter recombinant retrovirus containing the v-Ha-ras and v-myc oncogenes resulted in the apparent immortalization of a range of cell lines derived from 13- and 14-day murine fetal thymus, and expressing both v-Ha-ras and v-myc mRNA at high levels. Cell lines were established from untreated thymic lobes (group 1), deoxyguanosine-treated lobes (group 2), and in the presence of IL-2 (group 3). Cell lines with a wide range of surface phenotypes were established. Group 1 were adherent cell lines expressing many antigens, including Mac-1, FcR, and Ia. Group 2 were also adherent cells but expressed very few Ag. Group 3 were IL-2-dependent lymphoid-like non-adherent cells, with a null or early thymocyte-like phenotype. None demonstrated full rearrangement and expression of TCR alpha-, beta-, or gamma-genes, although cell lines in group 2 expressed short beta and those in group 3, short beta and gamma gene transcripts. This library of immortalized cell lines appears to represent several types of stromal cell and early thymocyte precursors present in 13- and 14-day fetal thymus. These cell lines should prove invaluable in reconstructing the environment of the intact fetal thymic lobe, to study the cellular interactions that appear to govern thymocyte proliferation, development, and selection of Ag reactivity.

Animals↗

PubMed1989

Density gradient profiles of A1 adenosine receptors labeled by agonist and antagonist radioligands before and after detergent solubilization.

A1 adenosine receptors in bovine cerebral cortex have been solubilized and subjected to sedimentation analysis using sucrose density gradient centrifugation. Because the receptors bound both agonists and antagonists with high affinity after solubilization, receptors labeled with an agonist or an antagonist radioligand could be studied before solubilization, after solubilization but before sucrose gradient centrifugation, or after sucrose gradient centrifugation. In each instance the agonist radioligand 125I-N6-p-aminobenzyladenosine (125I-ABA)-labeled receptor migrated as a single symmetrical peak that was located in the same area of the gradient. In contrast, the location of the receptor labeled with the antagonist [3H]xanthine amine congener [( 3H]XAC) varied in the different types of samples. When membranes were incubated with radioligands before solubilization, the peak of antagonist-labeled receptor was symmetrical and was located at a lower density than the peak of agonist-labeled receptor. In addition, receptors incubated with antagonist before solubilization migrated with an apparent lower density than receptors labeled with antagonist either after solubilization or after density gradient centrifugation. Treatments with agents that alter receptor/G protein interactions also resulted in a shift of antagonist-labeled receptors to lower density. These results suggest that the receptors that migrate to the lower density fractions of the gradients are free receptors, whereas those that migrate to the higher density fractions are coupled to a G protein. It is hypothesized that a large proportion of A1 receptors exist in the membrane coupled to a G protein and that this is the species labeled by the agonist radioligand 125I-ABA. It is, furthermore, hypothesized that the antagonist radioligand [3H]XAC preferentially binds to the free uncoupled A1 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗

PubMed1988

Effect of granulocyte-macrophage colony-stimulating factor and interleukin 3 on the v-src oncogene. Inhibition of tyrosine kinase activity in the absence of changes in gene expression.

Three cloned murine interleukin 3 (IL-3)-dependent cell lines have been converted to interleukin 2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF) growth-dependent states. FD.C/1 32Dcl-23 and GM cells grown and maintained as IL-3-dependent cell lines, and cells grown with GM-CSF have been infected with a murine recombinant retrovirus containing the v-src oncogene, and grown as lymphokine-independent cell lines. There is a significant increase in tyrosine kinase activity in cells which become lymphokine-independent. FD.C/1 and 32Dcl-23 cells maintained as IL-2-dependent cells lines and infected with the same virus did not grow as IL-2-independent cells. The lymphokine-independent cells FD.C/1src, 32Dsrc, and GMsrc all expressed high levels of tyrosine kinase activity, ranging from 5- to 20-fold more than levels measured in virus-infected cell lines maintained as IL-2-dependent cells. The exposure of FD.C/1src and 32Dsrc cells to IL-3, and GMsrc cells to IL-3 or GM-CSF, resulted in significant decreases in tyrosine kinase activity. These changes were rapidly reversed by removal of IL-3 or GM-CSF from these cells. However, the synthesis of v-src-specific RNA was not affected by the presence of IL-3 or GM-CSF in these cell lines. The biochemical pathways activated by IL-3 or GM-CSF inhibit the activity of the tyrosine kinase encoded by the v-src oncogene without altering gene transcription.

Animals↗

PubMed1988

Expression of functional human interleukin-2 receptors in murine interleukin-3-dependent cells.

A murine recombinant retrovirus containing the cDNA encoding the human p55 interleukin-2 (IL2)-binding protein was used to insert this gene into a murine interleukin-3 (IL3)-dependent cell line, FD.C/1. Virus-infected cells, maintained in medium supplemented with IL3, expressed human p55 on the cell surface and readily adapted to growth using human IL2. In the presence of human IL2, the synthesis of the endogenous murine p55 binding protein was induced in FD.C/1 cells, making it difficult to determine whether the human p55 protein was actively involved in the process of growth signal transduction. A cloned cell line, FD.huIL2R-2, was identified which grew in the presence of human IL2 but which had lost the ability to synthesize murine p55 protein. Growth of this clone was inhibited by the monoclonal antibody 2A3 which specifically blocked binding of human IL2 to the human p55 binding protein. Analysis of restriction enzyme digests of FD.huIL2R-2 cell DNA revealed that a rearrangement of a murine p55 gene had occurred, implying that virus infection had resulted in the integration of retroviral DNA at a site close to or within a murine p55 gene. If IL2 signal transduction involves binding to a surface heterodimeric receptor for IL2, it is argued that FD.huIL2R-2 cells contain an IL2 receptor complex of murine p70 and human p55 IL2-binding proteins. Alternatively, it is possible that integration of human p55 DNA into a site close to a murine p55 gene may lead to a hybrid p55 IL2-binding protein. If FD.huIL2R-2 cells express murine p70 IL2-binding protein as part of the receptor complex, the inability of cells to grow in murine IL2 implies that IL2 binding to p70 protein alone is insufficient for a growth signal in these cells. FD.huIL2R-2 cells grow at rates similar in IL3- or human IL2-dependent states. It is likely therefore that the biochemical pathways that control each of these lymphokine-dependent growth states are very similar.

Animals↗

PubMed1988

Characterization of cardiac A1 adenosine receptors by ligand binding and photoaffinity labeling.

[125I]N6-(p-aminobenzyl)adenosine and [125I]N6-(p-azidobenzyl)adenosine, which are potent agonists at A1 (Ri) adenosine receptors, have been used to characterize the adenosine receptor in membranes prepared from newborn chick heart. Scatchard analyses of [125I]N6-(p-aminobenzyl)adenosine binding to cardiac membranes revealed that the ligand bound to two affinity states of the receptor with Kd values of 0.7 and 9.9 nM. The corresponding maximum binding (Bmax) values were 25 and 86 fmol/mg of protein, respectively. In the presence of 0.1 mM 5'-guanylyl imidodiphosphate, a single affinity state was detected with a Kd of 9.4 nM and a Bmax of 96 fmol/mg of protein. Direct and indirect ligand binding studies with several adenosine receptor agonists and antagonists were used to compare the characteristics of the cardiac receptor with those of the A1 receptor in the cerebellum. The binding properties of the receptors in the two tissues were very similar although marked differences were observed in the binding kinetics of [125I]N6-(p-azidobenzyl)adenosine. Photo-affinity labeling experiments followed by sodium dodecyl sulfate-gel electrophoresis showed that the cardiac receptor had a apparent molecular weight of 37,600, which was slightly but significantly higher than that of the cerebellar receptor (35,500). The present results show that the cardiac receptor has ligand binding properties and a minimal subunit molecular weight similar to the more thoroughly studied A1 receptor in neural tissue.

Adenosine↗

PubMed1987

Correlation of agonist-induced phosphorylation of chick heart muscarinic receptors with receptor desensitization.

We have determined whether the process of agonist-mediated phosphorylation of the muscarinic receptor correlates with the process of muscarinic receptor desensitization in chick cardiac tissue. Exposure of ventricular slices to the agonist carbachol under conditions previously shown to lead to large increases in muscarinic receptor phosphorylation (Kwatra, M. M., and Hosey, M. M. (1986) J. Biol. Chem. 261, 12429-12432) resulted in decreased affinity of the muscarinic receptor for agonists. The agonist oxotremorine mimicked and the antagonist atropine prevented the effects of carbachol on receptor phosphorylation and agonist affinity. The time courses and concentration dependences for agonists to induce phosphorylation of the muscarinic receptor and decreases in agonist affinity were similar. Treatment of chick atria with acetylcholine under conditions which led to receptor phosphorylation resulted in decreased sensitivity of these preparations to the negative inotropic effect of carbachol. Taken together, the results support the concept that phosphorylation of cardiac muscarinic receptors may be related to the process of receptor desensitization. The mechanism by which agonists induce receptor phosphorylation was also investigated. The phosphorylated amino acids formed in response to agonists were serine and threonine. The protein kinase C activator phorbol myristate acetate had no effect on receptor phosphorylation or agonist affinity, nor did it prevent the effects of carbachol on either of these parameters. Receptor phosphorylation also was unaffected by the calmodulin antagonists W-7 and W-13, by elevation of cyclic nucleotides, and by agonists which activate other cardiac receptor systems. The results suggest that the phosphorylation of cardiac muscarinic receptors requires agonist occupancy of the receptor and/or may involve the participation of a selective protein kinase.

Animals↗

PubMed1987

A comparison of muscarinic acetylcholine receptors coupled to phosphatidylinositol turnover and to adenylate cyclase in guinea-pig atria and ventricles.

The affinities of muscarinic acetylcholine receptor agonist and antagonist compounds have been measured in studies of adenylate cyclase inhibition and phosphatidylinositol turnover in guinea-pig atria and ventricles. The affinities of all compounds tested were similar in atrial and ventricular preparations. However, three antagonists, atropine, pirenzepine and gallamine had different affinities at receptors coupled to adenylate cyclase inhibition compared with those which stimulated turnover of phosphatidylinositols. The neuromuscular blocking agents gallamine and pancuronium, which demonstrate cardioselective acetylcholine receptor antagonist activity, were competitive antagonists at receptors coupled to both second messenger systems. However, their affinities measured in both systems were lower than expected from tissue-response studies. Therefore, the cardioselectivity of these compounds cannot be explained by a simple competitive interaction with receptors coupled to either adenylate cyclase inhibition or phosphatidylinositol turnover.

Adenylyl Cyclases↗

PubMed1987

N6-phenyladenosines: pronounced effect of phenyl substituents on affinity for A2 adenosine receptors.

A number of N6-phenyladenosines with various substitutions on the phenyl ring have been synthesized and tested for their affinities toward brain A1 and A2 adenosine receptors. Compounds with meta substituents, such as (m-hydroxy- and m-iodophenyl)adenosine, were found to have high A1 selectivity. Meta substitution caused a selective decrease in the affinity of these compounds for A2 receptors. The results suggest that, in contrast to what is commonly held, certain N6-substituents have pronounced effects on affinity for brain A2 adenosine receptors. Thus, brain A2 receptors may have a well-defined region that recognizes N6-substitutions.

Adenosine↗

PubMed1986

Stimulation of phosphatidylinositol metabolism in atrial and ventricular myocytes.

Receptor-stimulated hydrolysis of inositol phospholipids was studied in atrial and ventricular myocytes isolated from guinea-pigs. Membrane phospholipids were labelled with [3H] inositol and their conversion to [3H] labelled inositol phosphate was measured in the presence of Li+ (10 mM). In the absence of added stimulatory hormones or neurotransmitters, little inositol phosphate accumulation was observed. Acetylcholine and carbachol stimulated inositol phosphate accumulation with a maximum of more than 12 times the unstimulated values in atrial myocytes and 7 times in ventricular myocytes. The EC50 values and 95% confidence limits for acetylcholine and carbachol were 0.9 microM (0.2 - 5.3) and 8.8 microM (6.3 - 11.8) in atria and 0.6 M (0.5 - 0.8) and 10.0 M (1.8 - 55.9) in ventricles, respectively. Oxotremorine was a partial agonist in stimulating inositol phosphate accumulation in both atrial and ventricular myocytes. The vasoactive peptides angiotensin II and vasopressin also stimulated inositol phosphate accumulation but the maximum effect was lower than that mediated through muscarinic receptors. However, the adenosine analogues, L-N6-phenylisopropyladenosine and 5'N-ethylcarboxamidoadenosine which, like muscarinic agonists depress cardiac contractility, did not affect inositol phosphate accumulation at concentrations up to 10(-4)M.

Acetylcholine↗

PubMed1986

Adenosine receptors in papilla of human kidneys.

Adenylate cyclase activity in homogenates of human papillae was stimulated by adenosine agonist compounds. The rank order of agonist potencies for stimulation was 5'-N-ethylcarboxamidoadenosine greater than L-N6-phenylisopropyladenosine greater than 2-chloroadenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine. This order of agonist potencies is similar to that observed at adenylate cyclase-coupled A2-adenosine receptors in many different tissues. The methylxanthines 8-phenyltheophylline and 1,3-diethyl-8-phenylxanthine acted as competitive antagonists, with 1,3-diethyl-8-phenylxanthine being approximately 10 times more potent than 8-phenyltheophylline. This study demonstrates the presence of A2-adenosine receptors in the papilla of human kidney. These receptors are similar to adenosine receptors previously observed in the renal papillae of the rat in terms of both agonist and antagonist potencies.

Adenosine↗

PubMed1986

Stimulation of phosphatidylinositol metabolism in the heart.

Receptor-stimulated hydrolysis of inositol phospholipids was studied in atrial and ventricular myocytes isolated from guinea-pigs. Acetylcholine and carbachol stimulated inositol phosphate accumulation with a maximum of more than 12 times the unstimulated values in atrial myocytes and 7 times in ventricular myocytes. The vasoactive peptides angiotensin II and vasopressin also stimulated inositol phosphate accumulation, but the maximum effect was lower than that mediated through muscarinic receptors. However, the adenosine analogues, L-N6-phenylisopropyladenosine and 5'N-ethylcarboxamidoadenosine which, like muscarinic agonists depress cardiac contractility, did not affect inositol phosphate accumulation at concentrations up to 10(-4) mol/l. Stimulation of phosphatidylinositol turnover in heart bears no obvious relationship to either contractility or release of atrial natriuretic factor.

Adenosine↗

PubMed1986

Effect of temperature on atrial and ventricular adenosine A1 receptors of the guinea pig.

Cardiac adenosine receptors are coupled to adenylate cyclase inhibition. In the guinea pig heart, the relative agonist potencies observed for adenylate cyclase inhibition were R-N6-phenylisopropyladenosine (R-PIA) = N6-cyclohexyladenosine greater than 5'-N-ethylcarboxamidoadenosine much greater than S-PIA. In both atrial and ventricular membranes, the antagonists 8-phenyltheophylline (8-PT) and isobutylmethylxanthine (IBMX) also showed similar affinities for atrial and ventricular adenosine receptors. The same pattern of relative agonist potencies was observed in experiments performed at either 25 or 37 degrees C. However, the maximal inhibition produced by R-PIA in atrial membranes decreased from 30.8 +/- 3.2% (n = 7) at 25 degrees C to 18.8 +/- 1.6% (n = 4) at 37 degrees C. No such difference in maximal inhibition was observed with ventricular membranes at these two temperatures (34.5 +/- 1.6%, n = 6 at 25 degrees C and 35.3 +/- 0.9%, n = 11 at 37 degrees C). While there was no change in agonist potencies, the affinities of the antagonists 8-PT and IBMX at cardiac adenosine A1 receptors were affected by temperature. At 25 degrees C, the pKD values for 8-PT and IBMX in ventricular membranes were 4.65 +/- 0.21 (n = 3) and 4.55 +/- 0.20 (n = 3), respectively. Their affinities were 7- to 19-fold higher at 37 degrees C, the pKD values being 5.93 +/- 0.12 (n = 7) (p less than 0.02) and 5.38 +/- 0.18 (n = 3) (p less than 0.05), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗

PubMed1985

Failure of gallamine and pancuronium to inhibit selectively (-)-[3H]quinuclidinyl benzilate binding in guinea-pig atria.

Binding of (-)-[3H]quinuclidinyl benzilate (QNB) to muscarinic sites in guinea-pig atrial and ileal longitudinal muscle homogenates showed the presence of a single population of binding sites in atria (KD = 41 (32-53) (95% confidence limits) pM; Bmax = 0.225 +/- 0.02 pmol/mg protein (3)) and two binding sites in the ileum (KD = 20.9 (8.8-49) pM and 11.3 nM; Bmax = 0.436 +/- 0.09 and 11.85 +/- 2.63 pmol/mg protein (4), respectively). Atropine, gallamine, and pancuronium displaced (-)-[3H]QNB binding from the high affinity binding sites in the two tissues in a dose-dependent manner with -log Ki values of 8.6, 6.4, and 6.9, respectively, in atria and 8.7, 6.8, and 6.9, respectively, in ileal longitudinal muscle. The lack of selectivity of gallamine and pancuronium in binding experiments differed from results obtained in isolated tissue experiments where these antagonists showed a marked difference in their ability to antagonize cholinomimetics in the two tissues. In addition, the Ki values for gallamine and pancuronium in ileal homogenates were ca. 130- and 16-fold lower, respectively, than their KB values determined from isolated tissue experiments. Attempts to correlate data from binding experiments and isolated tissue experiments using combinations of antagonists led to variable results attributed to differences in the rates of dissociation of the antagonists from muscarinic receptors. It is concluded that the interaction of gallamine or pancuronium with agonists or antagonists at muscarinic receptors is not a simple bimolecular interaction.

Animals↗

PubMed1984

Demonstration of RA - adenosine receptors in rat renal papillae.

Adenylate cyclase in homogenates of rat renal papillae was stimulated by adenosine agonist compounds. Adenosine agonists neither stimulated nor inhibited cyclase activity in cortex or medulla. The rank order of agonist potencies was 5'N- ethylcarboxamide adenosine much greater than 2-chloroadenosine greater than L-N6-phenylisopropyladenosine = cyclohexyladenosine greater than D-N6-phenylisopropyladenosine. Stimulation of adenylate cyclase by 5'N- ethylcarboxamide adenosine was competitively antagonised by the methylxanthines, 1,3-diethyl-8- phenylxanthine and 8-phenyltheophylline; 1,3-diethyl-8- phenylxanthine being approximately 10 times more potent than 8-phenyltheophylline. These results demonstrate adenosine receptors of the RA-type localized to the papilla of the rat kidney.

Adenosine↗

PubMed1983

Demonstration of adenylate cyclase coupled adenosine receptors in guinea pig ventricular membranes.

Adenylate cyclase in homogenates of guinea pig ventricles was inhibited by the stable adenosine analogs N6-phenylisopropyladenosine (PIA) and 5(1)-N-ethylcarboxamidoadenosine (NECA). Inhibition required GTP and was enhanced by sodium ion. The maximum inhibition observed was 35.1 +/- 1.1%, the EC50 (95% confidence limits, n) for PIA and NECA were 0.20 microM (0.17-0.25 microM, 6) and 0.66 microM (0.26-1.7 microM, 4) respectively. 8-Phenyltheophylline (10 and 100 microM) and isobutylemethylxanthine (100 microM) antagonized the inhibitory effects of the adenosine analogs. These results indicate that adenosine receptors of the inhibitory type (RI or A1) are present in guinea pig myocardium and may mediate some of the cardiac responses to adenosine.

Adenosine↗

PubMed1983

A simple technique to study the effect of drugs on the negative inotropic response elicited by postganglionic cholinergic nerve stimulation in guinea pig atria.

A simple technique is described for evaluating the effects of drugs on postganglionic cholinergic nerve stimulation in guinea pig atrial tissue. Raising the voltage of stimulation tenfold (high voltage stimulation, HVS) in the left atrium produced a positive inotropic response. Propranolol abolished this effect but failed to reveal a negative inotropic response. However, in atria obtained from 6-hydroxydopamine (6-OHDA) pretreated animals, HVS produced a negative inotropic effect. The magnitude of the response was frequency-dependent and was potentiated by the anticholinesterase dyflos. Tetrodotoxin abolished the response but mecamylamine had no significant effect. Drugs which inhibit cardiac muscarinic receptors caused a parallel shift of the frequency-response relationship. In the right atrium, HVS caused a biphasic inotropic response. Propranolol or 6-OHDA pretreatment inhibited the positive inotropic effect, thereby enhancing the negative inotropic response. The effect did not show as great a dependence on the frequency of stimulation as in the left atrium and was less useful for comparing the effectiveness of muscarinic receptor antagonists.

Animals↗