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J Mallol

Publications and source records attributed to J Mallol.

At least 73 records · Page 4Linked to original sources
PubMed1992

Optimal association-saturation procedure for estimating association and dissociation rate parameters in receptor studies. Application to solubilized A1 adenosine receptors.

A method of obtaining estimates of the maximum binding and association and dissociation rate constants for a receptor-ligand interaction is described. This new procedure, the association-saturation method, is based on an exact mathematical equation which defines the model without simplifications. The method proposed is readily applicable to any system consisting of one ligand and one receptor. With only four determinations, each involving one ligand concentration and an association time, it is possible to determine the number of specific binding sites, the dissociation and association rate constants and the equilibrium dissociation constant with accuracy. No dissociation curve is required, and only one point of the association curve for each ligand concentration is necessary. In addition to the higher confidence in the estimates of the parameter values obtained, this method leads to important savings in radiolabelled compounds and experimental time. The results were compared with those obtained with standard association-dissociation curves and saturation isotherms. The optimum number of replicates of each experimental point to obtain reliable estimates of different parameters is discussed on the basis of simulation studies. The performance of the procedure is analysed by means of association-saturation experiments with the agonist [adenine-2,8-3H,ethyl-2-3H]N6-phenylisopropyladenosine and solubilized A1 adenosine receptors from pig brain cortex.

Animals↗
PubMed1992

Modulation of adenosine agonist [3H]N6-(R)-phenylisopropyladenosine binding to pig brain cortical membranes by changes of membrane fluidity and of medium physicochemical characteristics.

The binding of [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) to pig brain cortical membranes was investigated as a function of membrane fluidity and of the physicochemical characteristics of the medium. The two affinity states of the A1 adenosine receptor behave differently in the experiments performed. Increases (up to 6.7 poises) or reduction (up to 1.3 poises) in membrane microviscosity did not lead to significant variation of equilibrium parameter values except for a slight modification of the low-affinity Kd value at 1.3 poises. Addition of cholesterol to raise the microviscosity up to 9.0 poises led to a marked decrease of binding to the high-affinity state without modifying the Kd. Furthermore, the Kd value for the low-affinity state rose markedly. Addition of sucrose, which modifies the conductance, viscosity and density of the aqueous medium, did not lead to significant changes when used at a concentration of 0.25 M, either in isolation or in radioligand binding assay medium. The presence of 0.32 M sucrose in the isolation medium led to a 30% fall in the total binding without affecting the distribution and Kd values for either affinity state very much. However, the presence of greater than or equal to 0.32 M sucrose in the binding assay medium produced the disappearance of the low-affinity states and the appearance of high-affinity states; only one affinity state was found to have a somewhat increased Kd value. On the other hand, increases of the medium conductance did not lead to the disappearance of the low-affinity state although some decrease of the binding was observed at high Tris concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The distribution of A1 adenosine receptor and 5'-nucleotidase in pig brain cortex subcellular fractions.

Pig brain cerebral cortex was subfractionated by isopycnic centrifugation in sucrose gradients. In each subfraction the content of the agonist [3H]R-PIA binding, the activity of adenosine metabolizing enzymes (5'-nucleotidase and adenosine deaminase) and the activity of membrane marker enzymes were determined. The fractions were also examined by electron microscope. In general, the results suggest a widespread distribution of A1 adenosine receptors in membranes from different origins. Marker enzyme profile characterization indicated an enrichment of A1 adenosine receptor in pre-synaptic membranes isolated from the crude synaptosomal fraction (P2B subfraction) as well as in membranes of glial origin such as myelin. The receptor is also present in the endoplasmic reticulum and in membranes isolated from the microsomal fraction that seem to have a post-synaptic origin (P3B). In subfractions having a high content of adenosine receptor the equilibrium binding parameters were obtained as well as the proportion of high- to low-affinity sites. From the values of the equilibrium constants it was not possible to find differences between the receptor in the different subfractions. Analysis of the affinity state distribution showed a diminished percentage of high-affinity sites in fraction P3A, which can be accounted by the existence of myelin membranes; in contrast the percentage of high-affinity states was higher in P2 and P3B, indicating that in these fractions the receptor is present in synaptosomal membranes. The close correlation shown between the enzyme 5'-nucleotidase specific activity and the specific ligand binding distributions led us to postulate an important role for the enzyme in the regulation of adenosine action in pig brain cortex.

5'-Nucleotidase↗
PubMed1992

The adenosine receptors present on the plasma membrane of chromaffin cells are of the A2b subtype.

The adenosine receptors in the plasma membrane of chromaffin cells from bovine adrenal medulla were characterized. The presence of A1 receptors was discounted owing to the absence of R-[3H]phenylisopropyladenosine (R-PIA) and [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) binding. The binding of the specific A2a ligand CGS-21680 was low. In contrast, the binding of 5'-(N-[3H]-ethylcarboxamidoadenosine ([3H]NECA) was relatively high (1.7 pmol/mg of protein at a ligand concentration up to 90 nM). This binding did not correspond to non-adenosine receptor NECA binding sites because the specific [3H]-NECA binding was similar when unlabeled adenosine, NECA, or R-PIA was used to measure the nonspecific binding. The rank order of potency of different ligands for the displacement of specific [3H]NECA binding was DPCPX greater than NECA greater than chloroadenosine greater than R-PIA greater than theophylline = CGS-21680. These results indicate that the receptors present on the plasma membrane of chromaffin cells are exclusively of the A2b subtype.

2-Chloroadenosine↗
PubMed1992

Characterization of adenosine receptors in brush-border membranes from pig kidney.

1. The adenosine receptors from pig kidney proximal tubules have been studied in membrane vesicle preparations derived from either luminal (brush-border membranes-BBM-) or basolateral (BL) sides. There was a substantial amount of A2-like NECA binding in both preparations, but the A1 subtype of adenosine receptors was not found in either BBM or BL membranes. The use of [3H]-CGS21680 which is a more specific ligand for A2a receptors revealed true adenosine receptors in the BBM. 2. The kinetic parameters for [3H]-CGS21680 binding to pig renal BBM were: Bmax = 1.48 pmol mg-1 protein and Kd = 150 nM. In the presence of Gpp(NH)p the affinity decreased (Kd = 220 nM), whereas the addition of Mg2+ induced a marked increase in affinity (Kd = 83 nM). These equilibrium constants are higher than those found for the A2a adenosine receptors present in pig brain striatal membranes (Kd = 12 nM), and are close to those found in rat renal BBM (Kd = 90 nM). 3. The order of potency of agonist and antagonists was not consistent with the presence of either A1 or A2 receptors, but it was very similar to the agonist order of potency for the A3 receptor subtype. Furthermore, the blockade of the [3H]-CGS21680 binding by both cholera and pertussis toxin further supports the view that the subtypes present in BBM are neither A1 nor A2. 4. Overall the results suggest the presence in BBM of an A3 receptor, or of a new subtype of adenosine receptor, which is linked to G proteins sensitive to both cholera and pertussis toxins.

Adenosine↗
PubMed1992

Characterization of the nuclear receptors of triiodothyronine (T3) by immunocytochemistry under electron microscopy.

By immunocytochemical methods, using colloidal gold particles coupled to antibodies, it is possible to label the occupied nuclear T3 receptors and observe them by electron microscopy. We carried out some experiments to validate these methods by testing the absence of non-specific binding of the antibodies to subcellular structures different from the T3 receptors. So, we have verified that the number of occupied T3 receptors in tissues from normal rats is lower in kidney cells than in heart and liver ones, a finding which is an agreement with data reported by others. The occupation of the receptors increases after T3 administration and decreases by triiodothyroacetic acid (Triac) administration to the rats, due to the displacement of T3 from the receptors with the latter. Our results are similar to those of the other authors using radioactive tracers. Non-specific binding was not detected. The occupied T3 receptors labelled with colloidal gold particles are located most commonly in the hetero/euchromatin interface. From our results we conclude that the colloidal gold particles observed by electron microscopy specifically label occupied T3 receptors, and the determination of the thyroid status at cellular level can be achieved using electron microscopy after labelling the receptors by immunocytochemistry with colloidal gold particles coupled to antibodies.

Animals↗
PubMed1991

N-ethylmaleimide affects agonist binding to A1 adenosine receptors differently in the presence than in the absence of ligand.

The effect of sulphydryl reagents (N-ethylmaleimide-NEM-4- hydroxymercuriobenzoate-HMB- and 5-5'-dithio-bis-2-nitrobenzoate-DTNB-) on agonist and antagonist binding to A1 adenosine receptors from pig brain was studied. The action of the mercurial agent HMB was found to be strong and seemed to be nonspecific. The effects of either NEM or DTNB were milder and more specific. The characterization of the agonist binding in membranes pretreated with moderate concentrations of DTNB and NEM led to reduced affinities for both high- and low-affinity sites without marked modifications of maximal binding or of proportion of affinity states. These results for NEM are surprising since the compound is usually used to mimick the effects of Gpp(NH)p, i.e. to shift high-affinity states to low-affinity states. It was found that this Gpp(NH)p-like effect of NEM is only possible when the compound is included in the assay medium. Similarly, Gpp(NH)p produces the uncoupling of the receptor molecule from G protein if included in the assay medium. Thus, membranes pretreated with Gpp(NH)p exhibited both affinity states and with similar equilibrium binding parameter values to those of the crude membranes.

Adenine↗
PubMed1991

The binding of [3H]R-PIA to A1 adenosine receptors produces a conversion of the high- to the low-affinity state.

Kinetic evidence for negative cooperativity on the binding of [3H]R-PIA to A1 adenosine receptors was obtained from dissociation experiments at different ligand concentrations and from the equilibrium isotherm. The dissociation curves indicate that there is an apparent ligand-induced transformation of high- to low-affinity states of the receptor. At concentrations of 18.2 nM R-PIA or higher there was only found the low-affinity state of the receptor. In view of these results equilibrium binding data were analyzed by the usual two-state model (assuming that there is an interconversion between them) and by the negative cooperativity model employing the Hill equation.

Animals↗
PubMed1991

Effect of phospholipases and proteases on the [3H]N6-(R)-phenylisopropyladenosine ([3H]R-PIA) binding to A1 adenosine receptors from pig cerebral cortex.

The effect of phospholipases and proteases on the membrane-bound and solubilized A1 adenosine receptor has been studied. Phospholipids modulate the [3H]N6-(R)-phenylisopropyladenosine binding to A1 adenosine receptors in crude membranes and in soluble preparations, because changes in the phospholipid environment decrease both the binding capacity and the affinity for the ligand. It has become clear that 1) there is co-solubilization of receptor and phospholipids; 2) the phospholipid requirements are different for the coupled and the uncoupled receptor; 3) a net charge in the polar head produced by phospholipase D prevents the agonist binding to the receptor-G protein complex; alternatively, when the whole polar head is removed by phospholipase C the uncoupled receptor is altered; and 4) the protease action upon the receptor suggests that receptor coupled to G protein is more protected by the membrane than the uncoupled receptor. In kinetic experiments performed on membranes it was demonstrated that phospholipase C and trypsin increased the Kd value of the high-affinity state by modifying both k1 and k-1. In contrast they only modified the dissociation constant of the low-affinity state. In conclusion it should be noted that phospholipids play a key role for the binding of R-PIA to A1 adenosine receptor. Also, a different disposition within the membrane of the coupled and uncoupled receptor is encountered.

Animals↗
PubMed1991

Air pollution and urinary thioether excretion in children of Barcelona.

The polluted environment found in highly industrialized areas and in big cities contains a great quantity of electrophilic (EC) and proelectrophilic (PEC) compounds, which largely contribute to the development of several pathological processes in humans. EC and PEC can be coupled to the cysteine moiety of glutathione spontaneously or by the glutathione S-transferase system (GST), giving nontoxic metabolites that can be eliminated as urinary thioethers (UT). Therefore one approach to establishing the degree of impact of EC and PEC on the human body is the analysis of UT in the population living in polluted environments. The work presented here has been carried out in the city of Barcelona with a group of 50 children living in a polluted area, over a 12-mo period. Our results demonstrate that UT are significantly higher when the amounts of air pollutants (AP) increase; although the level of contamination never exceeded the established "safe limits," UT reached values similar to those found in people smoking more than 10 cigarettes/d. These results may contribute to establishing the maximal levels of contamination compatible with a healthy life, on the basis of patterns of true salubrity rather than on political and economic criteria.

Air Pollutants↗
PubMed1991

Adenosine receptors in myelin fractions and subfractions: the effect of the agonist (R)-phenylisopropyladenosine on myelin membrane microviscosity.

In this article the existence of A1 adenosine receptors and the absence of A2 adenosine receptors in myelin membranes purified from pig brain white matter are demonstrated. The characterization of (R)-[3H]phenylisopropyladenosine ([3H]R-PIA) binding to purified myelin fractions was performed. The distribution of high- and low-affinity species of the A1 adenosine receptor was different in heavy, medium, and light myelin. The fluidity of myelin subfractions and of pig brain cortical membranes was estimated; the microviscosity of heavy myelin (5.4 poises) and of cortical membranes (5.1 poises) was similar and less than that of medium (7.8 poises) and light (8.2 poises) myelin. It was also demonstrated that the agonist R-PIA modifies the microviscosity of myelin membranes and that the degree of modification depends on the fluidity of the membrane assayed. These results suggest that adenosine receptors may have an important role in the functionality of myelin membranes.

Animals↗
PubMed1990

Glutathione-S-transferase activity in human superficial transitional cell carcinoma of the bladder. Comparison with healthy controls.

Glutathione-S-transferase (GST) activity and glutathione (GSH) content have been studied in human urinary bladder (UB) specimens obtained from healthy controls (HC) (n = 8) and from patients with superficial transitional cell carcinoma (TCC) (n = 9), either in TCC and in adjacent normal (ANE) tissues of the same patient. The GST activity was significantly higher in TCC in comparison with ANE (ten fold) and with HC (five fold). This activity was also significantly higher in HC than in ANE (two fold). The Km values obtained in the whole population (1.26 +/- 0.3 X 10(-3) mol/l) suggest that a unique form of isoenzyme is present in the UB epithelium and that it is the same acidic form "rho" described in erythrocytes. The GSH content was significantly higher in TCC than in ANE (2.5 fold) and also that in HC (three fold). A good correlation between GST activity and GSH content was observed in HC but not in TCC or ANE. These results demonstrate the relation between the activity of the GST system and the development of the TCC as well as its role in the cellular resistance to chemotherapy. A possible decrease of the GST activity before the development of the tumor is also discussed.

Aged↗
PubMed1990

Adenosine metabolism in kidney slices under normoxic conditions.

The effect of adenosine in rat kidney under normoxic conditions has been studied. It is demonstrated that adenosine modulates cell nucleotide levels. HPLC analysis of the purine compounds inside the cell indicates that adenosine improves the ATP/ADP ratio, whereas it diminishes the adenine content. This behaviour is not due to mediation by specific receptors, as agonists at P1 purinoceptors did not have any effect. Further evidence using inosine as well as dipyridamole and deoxycoformycin indicates that all effects are dependent on the previous uptake of adenosine. The origin of free adenine in the kidney has been investigated, and it appears to come from the phosphorolysis of 5'-methylthio-adenosine. This report is the first to describe the activity of methylthioadenosine phosphorylase (E.C. 2.4.2.28) in the kidney. It is concluded that 1) extracellular adenosine improves guinea pig renal function by increasing the ATP level and the ATP/ADP ratio; and 2) there exists a functional pathway in the kidney that produces adenine and AMP coming from methionine and ATP. This latter pathway probably produces spermine and spermidine, which are likely to be important for renal function.

Adenine↗
PubMed1990

Solubilization of A1 adenosine receptor from pig brain: characterization and evidence of the role of the cell membrane on the coexistence of high- and low-affinity states.

The present solubilization strategy recognizes the important role of detergent cocktails in the solubilization and subsequent stability of adenosine A1, receptors from pig brain cortical membranes. The 3-[3-(cholamidopropyl)dimethylammonio]-1-propane-sulfonate-digitonin mixture produced the extraction of up to 52% of the receptor with an enrichment of 1.2-fold with respect to crude membranes. The binding activity of the soluble extract was very stable even in the absence of glycerol. In crude membranes the existence of high- and low-affinity states was detected, but in the soluble extract and in the detergent-treated membranes only the high-affinity state was detected. Association-dissociation curves showed that in crude membranes no interconversion between high- and low-affinity sites is produced by the association of the ligand [3H]R-N6-phenylisopropyladenosine. These results suggest that the high- and low-affinity states are different conformations induced by the structure of the membrane. The modulation of the binding activity by (Gpp(NH)p) 5'-guanylylimidodiphosphate and Mg2+ was studied. In crude membranes Gpp(NH)p shifted the high-affinity state to the low-affinity state, whereas the contrary occurred when Mg2+ was used. The effect of both Mg2+ and Gpp(NH)p was also assayed with the soluble extract and with the detergent-treated membranes. In addition to a decrease of the overall binding capacity, Gpp(NH)p promoted a conversion to all low-affinity states in the detergent-treated membranes or to all very-low-affinity sites in the soluble extract. Mg2+ and Gpp(NH)p counteracted their effects in intact membranes, whereas Mg2+ could not reverse the uncoupling effect of Gpp(NH)p with solubilized or detergent-treated membranes. Thus, it is suggested that Mg2+ acts at sites other than guanine-nucleotide-sensitive sites. If high-affinity states correspond to receptor/G protein complexes and low-affinity states correspond to the uncoupled receptor, we should conclude that Mg2+, as well as the loss of membrane integrity, favours the interaction of A1 receptor molecule with G protein.

Animals↗
PubMed1990

Quantum chemical study of the electronic and conformational characteristics of adenosine and 8-substituted derivatives: functional implications in the mechanism of reaction of adenosine deaminase.

A quantum chemical study of 10 substrates of adenosine deaminase is performed. The conformational preference around the glycosidic bond of several 8-substituted derivatives of adenosine is studied using semiempirical modified neglect of diatomic overlap (MNDO) and Austin model 1 (AM1) methods. All the compounds studied show preference for the anti conformation; the syn - anti energetic differences calculated are small and in excellent agreement with experimental data. A relationship between the ab initio molecular electrostatic potential minimum energy of N3 and the syn - anti energetic difference is found. A highly significant relationship is also found between the ab initio net charge over the purine and pyrimidine rings and the logarithm of the maximum rate of deamination (log Vm) of the nucleosides by adenosine deaminase. In contrast, no significant relationship is found between the anti preference of 8-substituted derivatives of adenosine and their log Vm of deamination.

Adenosine↗
PubMed1990

A method for binding parameters estimation of A1 adenosine receptor subtype: a practical approach.

Working with pig brain striatum in which A1 and A2 adenosine receptor subtypes coexist, we describe an uncomplicated method for unequivocally obtaining the equilibrium parameters (KD and binding capacity) of A1 receptor without interference from ligand binding to A2 receptor. Also, the equilibrium parameter estimation method we propose avoids the experimental determination of nonspecific binding by the inclusion of the corresponding unknown parameter in the function. This not only saves time but also avoids the use of expensive radioligands in saturation experiments. The method is suitable for any system with two different receptor subtypes for the same physiological ligand, and good estimates of the equilibrium parameters corresponding to the subtype displaying the higher affinity for the ligand can be obtained.

Adenosine↗