Biomedical subjects

J F Rodrigues de Miranda

Publications and source records attributed to J F Rodrigues de Miranda.

At least 19 recordsLinked to original sources

PubMed1999

A molecular model for the synergic interaction between gamma-aminobutyric acid and general anaesthetics.

Within the context of the discussion about rational polytherapy, we determined the effects of four anaesthetics on the binding of [3H]t-butylbicycloorthobenzoate ([3H]TBOB) to the GABA(A) receptor complex in the presence of several concentrations of GABA (gamma-aminobutyric acid), in order to build a molecular model that can describe and quantify the interactions between the compounds. The empirical isobole method revealed that GABA and the anaesthetics acted synergically in displacing [3H]TBOB. This synergy could be described by a simple molecular model in which both GABA and the anaesthetics displaced [3H]TBOB allosterically and in which GABA allosterically enhanced the binding of the anaesthetics. To get information about the interaction between GABA and anaesthetics, we used [3H]TBOB as a tracer ligand. The model indicated that GABA enhanced the affinity of thiopental 3.0-fold, propofol 5.0-fold, the neuroactive steroids Org 20599 3.5-fold and Org 20549 13-fold. Insight into the molecular mechanism and strength of these interactions can help clinicians to choose therapeutically optimal drug and dose combinations: a step towards rational polytherapy.

Allosteric Site↗

PubMed1995

Pancuronium masks the prejunctional muscarinic autoreceptor in guinea pig tracheal smooth muscle.

The effect of pancuronium pretreatment on the function of the prejunctional muscarinic receptor in guinea-pig trachea was studied by using electrical field stimulation (EFS). The effects of cumulative doses of the muscarinic M2 receptor antagonist gallamine were investigated in tracheal smooth muscle strips from guinea-pigs after addition of pancuronium in vitro and in strips from guinea-pigs which had been pretreated with doses of pancuronium that caused 100% neuromuscular blockade. The results of both types of experiments were compared to those of control groups of the same size. In all strips a dose response curve with cumulative doses of methacholine was made before EFS was switched on. No differences were found between the mean pD2 value and slope of the concentration-response curves of untreated guinea-pigs and animals treated with anaesthetics and pancuronium. The animals showed variable responses to pancuronium. The bath concentration of pancuronium which decreased the EFS-induced contraction to half the original value varied between 14-61 microM. The intravenous dose necessary to paralyze the muscles, varied among the different guinea-pigs from 0.017-0.085 mg.kg-1. The EFS-induced contraction for the concentration range of gallamine 0.32 microM-0.32 mM was found to differ significantly between the strips treated with pancuronium in the organ bath and their control group. For the guinea-pigs anaesthetized and pretreated with pancuronium a significant difference with control was observed at gallamine concentrations ranging from 0.032-0.32 mM. These results show that pancuronium, added to the organ bath as well as administered intravenously to the guinea-pig, masked the inhibitory muscarinic receptor.

Animals↗

PubMed1994

Binding and functional properties of hexocyclium and sila-hexocyclium derivatives to muscarinic receptor subtypes.

1. We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB-OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors. 2. Sila-substitution (C/Si exchange) of hexocyclium (-->sila-hexocyclium) and demethyl-hexocyclium (-->demethyl-sila-hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila-substitution of o-methoxy-hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied. 3. The p-fluoro- and p-chloro-derivatives of sila-hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies. 4. In binding studies, o-methoxy-sila-hexocyclium (M1 = M4 > or = M3 > or = M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o-methoxy-sila-hexocyclium for the prejunctional M1/M4-like heteroreceptors in rabbit vas deferens. 5. The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-sila-hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives.

Animals↗

PubMed1993

Characterization of muscarinic receptors in rat kidney.

Muscarinic receptors in mammalian kidney seem to be involved in diuresis. In this study we give a detailed characterization of receptors in rat kidney. Specific binding of [3H](-)-quinuclidinylbenzilate ([3H]QNB) to membranes of rat kidney cortex was saturable and of high affinity. A dissociation constant of 0.063 +/- 0.003 nM and a receptor density of 1.46 +/- 0.07 pmol/g wet weight were obtained. The dissociation kinetics could be best described by assuming a mono-exponential function (k-1 = (0.52 +/- 0.1) x 10(-4) s-1). The binding of [3H]QNB reached a maximum in 60 min at 0.6 nM at 37 degrees C. Competition experiments with the enantiomers of benzetimide confirmed the muscarinic nature of the [3H]QNB binding sites. The inhibition constants of pirenzepine (0.23 +/- 0.02 microM), (+-)-hexahydrosiladifenidol (0.040 +/- 0.002 microM), AF-DX 116 (1.45 +/- 0.07 microM), methoctramine (1.67 +/- 0.02 microM) and gallamine (78 +/- 3 microM) classified this receptor as an M3 receptor. Inhibition of [3H]QNB binding by the agonists methylfurtrethonium, arecoline, isoarecoline methiodide, arecaidine propargyl ester and McN-A-343 displayed monophasic inhibition curves. With (+/-)-cis-2-methyl-4-dimethylaminomethyl-1,3- dioxolane methiodide in two out of four experiments a small (11%) population of high affinity agonist sites could be detected. The potassium sparing diuretic amiloride inhibited [3H]QNB binding (36 +/- 3 microM). Although in a way related to the amiloride binding site, the muscarinic receptors in rat kidney are unlikely to be the primary target of diuretic action of this drug.

Amiloride↗

PubMed1992

Does treatment with beta-adrenergic blocking agents cause a decrease in beta 2-adrenoceptor affinity?

The effect of beta-adrenoceptor antagonists (BAAs) differing in lipophilicity and partial agonist activity (PAA), and a full agonist, on the dissociation constant for [125I]-(-)- iodocyanopindolol binding to beta 2-adrenoceptors (KD) has been investigated. Twelve healthy, normotensive male volunteers (mean age 22.3 y) were treated with different BAAs according to a cross-over design. The drugs used were propranolol (highly lipophilic BAA, no PAA), pindolol (moderately lipophilic BAA, strong PAA), dilevalol (highly lipophilic BAA, weak PAA) and salbutamol (full agonist). Before and after a single dose and an 8 day course of one of the drugs, blood pressure and the beta 2-adrenoceptor characteristics of mononuclear leukocytes (MNL) were determined. Between the treatment periods, there was a washout interval of 14 days. All BAAs decreased the blood pressure, but only propranolol lowered heart rate. Treatment with salbutamol decreased the diastolic and increased the systolic blood pressure and heart rate. Three hours after the single dose of any of the BAAs, a more than 2-fold increase in KD was observed, and the increase became larger after 8 days of administration (up to 3.7-fold increase). In contrast, no effect on KD was observed after treatment with salbutamol. BAAs with PAA and salbutamol induced a 30% decrease in beta 2-adrenoceptor density. It is concluded that treatment with BAAs, irrespective their lipophilicity or PAA, induces a decrease in the affinity of MNL beta 2-adrenoceptors for antagonists. This phenomenon may help to explain the contradictory relationship between the kinetics and dynamics of BAAs.

Adrenergic beta-Antagonists↗

PubMed1992

The GABAA receptor complex in relation to epilepsy. Reversal of [3H]TBOB inhibition: a prediction of proconvulsive properties?

[3H]-t-Butylbicycloorthobenzoate ([3H]TBOB), a convulsant, is known to label a binding site on the GABAA receptor complex. Bicuculline methochloride (bicuculline MCl), folic acid, pentazocine, naloxone, ethyl-beta-carboline-3-carboxylate (beta CCE) and Ro 5-4864 have (pro)convulsive properties in vivo. In the present study, we determined the extent to which these compounds modify the binding of [3H]TBOB in the presence of IC50 amounts of GABA (5 microM) or diazepam (50 microM). We found that the GABA antagonist bicuculline MCl reversed the inhibitory effect of GABA on [3H]TBOB binding completely, as was expected. Folic acid, pentazocine and naloxone also reversed the inhibitory effect of GABA on [3H]TBOB binding. This finding is compatible with the view that the proconvulsive effects of these compounds can be credited to a reduction of GABAergic action at the GABAA receptor complex. We suggest that the reversal of GABA's inhibition of [3H]TBOB binding is a sufficient (but not a necessary) condition to predict proconvulsive (side) effects of drugs. beta CCE and Ro 5-4864 modified [3H]TBOB binding in the presence of GABA in a biphasic fashion. A unique relation between beta CCE, Ro 5-4864 and the GABAA complex might exist. Bicuculline MCl reversed the inhibitory effect of diazepam on [3H]TBOB binding only partly. beta CCE did not reverse the inhibitory effect of diazepam on [3H]TBOB binding, neither did Ro 5-4864. The presence of a GABA-independent interaction between a low affinity benzodiazepine recognition site and the TBOB site is proposed.

Animals↗

PubMed1991

Selective labelling of muscarinic M1 receptors in calf superior cervical ganglia by [3H](+/-)-telenzepine.

A method was developed to determine the affinities of antimuscarinic drugs at M1 receptors. [3H](+/-)-Telenzepine served as radioligand in crude preparations of calf superior cervical ganglia and showed high affinity for a single receptor population, consisting of M1 receptors (KD = 1.12 nM). Kinetic experiments showed monophasic association (k1 = 0.017 min-1 nM-1) and dissociation (k-1 = 0.017 min-1) kinetics, the half-life of dissociation being 41 min at 37 degrees C. The kinetic KD value amounted to 1.00 nM. M1 affinities for pirenzepine, methoctramine, hexahydro-sila-difenidol and p-fluoro-hexahydro-sila-difenidol determined in competition experiments were similar to those found in functional studies with M1 receptors in rabbit isolated vas deferens. The binding assay was used to determine the affinities of the (R) and (S) enantiomers of tertiary (trihexyphenidyl, hexahydro-difenidol, hexbutinol, p-fluoro-hexbutinol) and quaternary muscarinic antagonists (trihexyphenidyl methiodide, hexbutinol methiodide). Comparison of results obtained with the rabbit vas deferens suggested that the ionic environment may influence the affinities.

Animals↗

PubMed1991

Prejunctional muscarinic receptors on cholinergic nerves in guinea pig airways are of the M2 subtype.

Prejunctional inhibitory muscarinic receptors in guinea pig tracheal strips were investigated by electrical field stimulation. Pilocarpine and methacholine caused, in a similar way, a dose-dependent increase in baseline with a concomitant decrease in twitch response. We showed by using selective muscarinic antagonists, such as pirenzepine (M1-selective), methoctramine (M2-selective), AF-DX 116 (11-[[2-[diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro- 6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one, M2-selective), gallamine (M2-selective) and 4-DAMP (4-diphenylacetoxy-N- methylpiperidinemethiodide, M3-selective), that the prejunctional inhibitory muscarinic receptor is of the M2 subtype.

Animals↗

PubMed1991

Alterations of muscarinic acetylcholine receptors in the nasal mucosa of allergic patients in comparison with nonallergic individuals.

Cholinergic nasal hyperresponsiveness in nasal allergy may be due to changes of the characteristics in muscarinic cholinergic receptors. Radioligand receptor binding and in vitro autoradiographic studies of nasal mucosa in nonallergic (NA) and allergic patients were performed to investigate this hypothesis. The heterogeneous NA group was subdivided into control individuals and patients with chronic sinusitis and vasomotor rhinitis. The 3H-(-)-Quinuclidinylbenzilate binding to muscarinic receptors in human nasal mucosa membranes was saturable and of high affinity in all groups. No significant differences could be demonstrated between the subgroups of the NA patients. In allergic patients the dissociation constants and receptor densities were significantly decreased in comparison with those of NA and with those of control individuals. No differences in agonist binding or coupling of the muscarinic receptor to the effector system via the G protein could be observed in allergic patients. In vitro autoradiographic experiments demonstrated specific 3H-(-)-Quinuclidinylbenzilate labeling of the glandular acini in NA and allergic patients. No specific labeling could be observed in the epithelium, blood vessels, or connective tissue. In conclusion, the increased sensitivity and decreased muscarinic receptor number may reflect the cholinergic-induced hypersecretion in nasal allergy but are probably too small to explain the complex allergic reaction.

Acetylcholine↗

PubMed1991

Alterations of adrenoceptors in the nasal mucosa of allergic patients in comparison with nonallergic individuals.

Nasal hyperreactivity in nasal allergy may be due to changes of the characteristics in adrenergic receptors. Radioligand receptor-binding studies with the antagonists, 3H-prazosin (alpha 1-adrenoceptor), 3H-rauwolscine (alpha 2-adrenoceptor), and 125I-(-)-Cyanopindolol (beta-adrenoceptor) were performed in homogenates of nasal mucosa of allergic and nonallergic (NA) patients to investigate this hypothesis. The heterogeneous NA group was subdivided into control individuals and patients with chronic sinusitis and vasomotor rhinitis. No significant differences in affinities or densities of alpha 1- and alpha 2-adrenoceptors could be demonstrated in allergic patients in comparison with NA and control individuals. The beta-adrenoceptor density was significantly reduced in allergic patients in comparison with that of control individuals. Neither changes in agonist binding or in the effect of Gpp(NH)p on the agonist binding to beta-adrenoceptors could be observed in allergic patients. The subtype selective antagonist, LK203-030, demonstrated the presence of a homogeneous population of beta 2-adrenoceptors in human nasal mucosa of both NA and allergic patients. In vitro, autoradiography demonstrated specific 125I-(-)-Cyanopindolol labeling of the epithelium in NA and allergic patients. In conclusion, no changes in characteristics of alpha 1- or alpha 2-adrenoceptors in the nasal mucosa could be demonstrated in nasal allergy. However, a decreased number of beta-adrenoceptors may reflect a beta-adrenergic abnormality in nasal allergy.

Autoradiography↗

PubMed1991

Adrenoceptors on blood cells from patients with primary Raynaud's phenomenon.

1. alpha 2-Adrenoceptors on platelet membranes and beta 2-adrenoceptors on lymphocytes were studied in 24 patients with primary Raynaud's phenomenon and in 24 age- and sex-matched control subjects. In two subgroups, a standardized mental arithmetic test and a finger-cooling test were performed. 2. Baseline blood pressure, heart rate and forearm blood flow did not differ between the two groups. 3. Baseline skin microcirculation (laser Doppler flux) was decreased in primary Raynaud's phenomenon (19 +/- 15 arbitrary units) compared with control subjects (33 +/- 14 arbitrary units) (P less than 0.01). 4. Baseline plasma noradrenaline concentration (2.00 +/- 1.44 versus 1.16 +/- 0.36 nmol/l) and alpha 2-adrenoceptor density (301 +/- 119 versus 210 +/- 82 fmol/mg) were increased in patients with primary Raynaud's phenomenon in comparison with the control subjects. The alpha 2-adrenoceptor density/beta 2-adrenoceptor density ratio in patients with primary Raynaud's phenomenon was, with a value of 0.37 +/- 0.04, higher than in the control subjects, where a value of 0.25 +/- 0.02 was measured (P less than 0.001). Plasma adrenaline concentration, beta 2-adrenoceptor density and the antagonist affinity to both receptor subtypes did not differ between both groups under baseline conditions. 5. Whereas during the finger-cooling test no differences were seen in the responses of the parameters measured, the mental arithmetic test induced an increase in laser Doppler flux in patients with primary Raynaud's phenomenon and a decrease in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1991

Binding of Ro 5-4864 to brain membranes of rats with and without absence-like phenomena.

Spontaneously occurring spike-wave discharges (SWD) in rats are used as a model for absence epilepsy in humans. In vitro, the binding parameters of 3H-Ro 5-4864, a ligand labelling the peripheral benzodiazepine receptor, were determined for brain membranes of WAG/Rij rats, an inbred strain showing SWD, and for ACI rats, an inbred strain showing no SWD. No difference in the Kd but a small difference in the Bmax values between the strains were found. Recently, other investigators reported a correlation between a decrease in affinity for 3H-Ro 5-4864 and the occurrence of SWD. Our results suggest however that it is doubtful that a change in Kd of the peripheral benzodiazepine receptor is causal in the etio-pathology of the spontaneous absence like phenomena in rats.

Animals↗

PubMed1990

Biochemical and autoradiographic analysis of beta-adrenoceptors in rat nasal mucosa.

The specific binding of 125I-(-)-cyanopindolol (125I-(-)-CYP) to homogenates and cryostat sections of rat nasal mucosa was saturable, stereoselective and of high affinity (Kd = 5.0 +/- 0.4 pM. Bmax = 204 +/- 12 fmol/mg protein and Kd = 7.2 +/- 0.7 pM; Bmax = 15 +/- 1 fmol/mg protein respectively). The subtype-selective antagonists, LK203-030 and ICI118,551, inhibited specific 125I-(-)-CYP binding according to a two-binding site model, indicating the presence of 57 and 45% beta 1-adrenoceptors in homogenates and cryostat sections, respectively. Competition of isoprenaline for antagonist binding to homogenates demonstrated 30 +/- 3% high-affinity agonist binding sites. A steepening of the curve was observed in presence of guanine nucleotides. In vitro labelling of cryostat sections of rat nasal mucosa was combined with autoradiography. The autoradiographs generated after incubation with 20 pM 125I-(-)-CYP showed specific labelling of the epithelium and glandular excretory ducts. It appeared from autoradiographs generated with subtype-selective antagonists in addition to the radioligand that beta 1- and beta 2-adrenoceptors were present in both structures.

Animals↗

PubMed1990

Alpha 2-adrenoceptors in homogenates of rat nasal mucosa.

The specific binding of [3H]rauwolscine to rat nasal mucosa membranes was saturable, stereoselective and of high affinity. The Scatchard plot pointed to a homogeneous population of binding sites (Kd = 3.6 +/- 0.6 nM; Bmax = 5.1 +/- 0.7 pmol/g). The non-specific binding appeared to be non-linear, probably due to filter binding. Inhibition of [3H]rauwolscine binding with the subtype-selective antagonist, prazosin, suggested the presence of alpha 2-adrenoceptor subclasses in rat nasal mucosa. The (-)-epinephrine inhibition curves demonstrated high- and low-affinity agonist binding sites. A monophasic (-)-epinephrine inhibition curve was obtained in the presence of guanine nucleotides.

Animals↗

PubMed1990

Binding of the cage convulsant, [3H]TBOB, to sites linked to the GABAA receptor complex.

[3H]t-Butylbicycloorthobenzoate ([3H]TBOB) binds to specific sites on crude synaptic rat brain membranes. The dissociation constant, Kd, determined from saturation experiments is near 8 nM and the receptor density Bmax is about 20 pmol/g wet tissue. Non-specific binding constitutes about 35% of the total binding at 4 nM [3H]TBOB. The association of [3H]TBOB is monophasic but its dissociation is biphasic. Kd values of 8 nM (70% of the binding sites) and 20 nM (30% of the binding sites) were estimated from the kinetic data. These values differ from those previously reported. Specifically bound [3H]TBOB is displaced by picrotoxin and by t-butylbicyclophosphorothionate (TBPS). No simple competitive interaction of picrotoxin with [3H]TBOB binding was found. Micromolar quantities of the GABAergic facilitating compounds, GABA, muscimol and diazepam inhibited [3H]TBOB binding in an allosteric manner.

Animals↗

PubMed1990

Pharmacokinetic properties of the antimuscarinic drug [3H]-hexahydro-sila-difenidol in the rat.

The pharmacokinetics of tritiated hexahydro-sila-difenidol [( 3H]-HHSiD) were examined in rats. Furthermore, the distribution of radioactivity was studied by means of whole body autoradiography. After i.v. administration of 2.9 mg/kg HHSiD plus [3H]-HHSiD to anaesthetized rats bearing a catheter implanted in the ductus choledochus and receiving a mannitol infusion, HHSiD was rapidly distributed and metabolized. Only 5% of the radioactivity was recovered in blood after 23 s and 0.4% after 2.5 h. 64% of the plasma radioactivity could be extracted with hexane from the samples taken 23 s after administration. 52% of the radioactivity was eliminated within 2.5 h, 13% by urinary and 39% by biliary excretion. Following oral administration of 8.6 mg/kg HHSiD plus [3H]-HHSiD there was an absorption of approximately one fourth of the administered radioactivity within 4 h. By means of whole body autoradiography (i.v. injection) as well as by tissue distribution measurement the highest levels of radioactivity were found in bile, urine, lung, kidney, adrenals, liver and pancreas. Thus, after i.v. administration to rats HHSiD is rather quickly distributed, metabolized and excreted. This explains its low antimuscarinic potency in vivo.

Administration, Oral↗

PubMed1990

Folates: epileptogenic effects and enhancing effects on [3H]TBOB binding to the GABAA-receptor complex.

The biochemical mechanism responsible for the convulsive effects of folates was investigated. The epileptogenic effects of folates were determined in vivo by quantification of the seizures following intracortical application in rats. The rank order of epileptogenic effects is: folic acid greater than or equal to 5-HCO-H4 folate greater than H2 folate greater than 5-CH3-H4 folate. This sequence of epileptogenicity in vivo is compared to the rank order of the effects of folates on radioligand binding to the GABAA-receptor complex in vitro. The inhibitory potencies of folates on [3H]muscimol and [3H]diazepam bindings did not correlate with their epileptogenic effects. However, folates reverse the inhibiting effect of GABA on the binding of the cage convulsant [3H]TBOB [( 3H]t-butylbicycloorthobenzoate). The rank order of this in vitro effect (folic acid greater than 5-HCO-H4 folate greater than H2 folate = 5-CH3-H4 folate) resembles the rank order of epileptogenicity determined in vivo. A relationship between the in vivo and in vitro effects is therefore suggested.

Animals↗

PubMed1990

Beta 2-adrenoceptor up-regulation in relation to cAMP production in human lymphocytes after physical exercise.

The effect of isotonic bicycle exercise on beta 2-adrenoceptors and cAMP production in lymphocytes was studied. The origin of the newly exposed receptors was studied by measuring radioligand binding in the presence of hydrophylic or lipophylic 'cold' ligands. During exercise beta 2-adrenoceptor density increased from 1207 +/- 144 to 1776 +/- 152 sites/cell (56 +/- 14%). Isoprenaline induced an increase in cAMP synthesis, which was 68 +/- 15% higher than those values measured before the test. Forskolin did not induce a higher increase in cAMP production after exercise. The increase in receptor density seems not to be due to a transfer of internalized receptors to the membrane surface. We conclude that physical exercise induces an up-regulation of beta 2-adrenoceptors on lymphocytes and that these adrenoceptors are functional.

Adult↗