Autoradiographic localisation of ascorbic acid-dependent binding sites for [125I]iodocyanopindolol in guinea-pig trachea

Light microscopic autoradiography showed that the supposedly beta-adrenoceptor-selective radioligand [125I]iodocyanopindolol (I-CYP) bound to sites in both the guinea-pig tracheal epithelium and smooth muscle that were sensitive to propranolol and isoprenaline. Low levels of binding were associated with sub-epithelial mucosal cells. Ascorbic acid caused a concentration-related increase in total I-CYP binding which was predominantly associated with the sub-epithelial mucosa, was not inhibited by propranolol, and was thus not associated with beta-adrenoceptors.     

Eosinophil peroxidase and 125I- ion binding in guinea-pig trachea.

The present study further characterizes ascorbic acid-dependent 125I- ion binding in guinea-pig trachea. Binding of 125I- ion in the presence of ascorbic acid was detected at the epithelial/submucosal interface, apparently involving individual cells containing peroxidase enzyme. A similar binding pattern was observed when hydrogen peroxide was substituted for ascorbic acid. Binding could be inhibited by the addition of the H2O2 degrading enzyme catalase or the peroxidase inhibitor thiourea. Results demonstrated that this binding was dependent upon the addition of, or endogenous production of hydrogen peroxide and its metabolism via airway eosinophil peroxidase. The relevance of this anomalous iodine binding phenomenon to radioiodinated ligand binding studies is discussed.     

N-Acetylcysteine and mucociliary activity in mammalian airways

The effect of N-acetylcysteine on mucus trasnport velocity (MV), ciliary beat frequency (CBF), mucus production (MP), mucus lysis and on the micro-morphology of the secretory cells was studied in mammalian airways. The results showed that: 1. MV increased in healthy rats and rabbits, as well as in bronchitic rats, after concentrations as low as 10(-14) g/ml. Depression of MV occurred first at 10(-6) and 10(-5) g/ml in healthy and bronchitic animals, respectively. 2. CBF was stimulated at concentrations between 10(-12) and 10(-10) g/ml and decreased at concentrations above 10(-8) g/ml. 3. MP increased by approximately 100% over control values. 4. Lysis of stagnant mucus was evident first at a concentration of 10(-11) g/ml after 15 min incubation. 5. TEM confirmed the increased activity of the mucus secreting cells and showed that no pathological changes occurred within the cell following incubation at 10(-7) g/ml for up to 150 min. The importance of these findings on the overall mucociliary function is discussed.     

Characterization of [125I]-endothelin-1 and [125I]-BQ3020 binding to rat cerebellar endothelin receptors.

1. We performed radioligand binding experiments on rat cerebellar homogenates using [125I]-endothelin-1 ¿[1251]-ET-1¿ and [125I]-BQ3020 to examine the pharmacology of endothelin receptors in rat brain. Saturation experiments demonstrated a single population of binding sites with high affinity for both radioligands ([125I]-ET-1, pKd = 8.94 +/- 0.17; [125I]-BQ3020, pKd = 9.18 +/- 0.14 nM; mean +/- s.e.mean). However, [125I]-BQ3020 only recognised approximately one third the number of endothelin receptors measured with [125I]-ET-1. 2. Saturation binding experiments with [125I]-PD151242 revealed high affinity binding to a single population of ETA receptors in the cerebellar homogenates (pKd = 9.95 +/- 0.14; Bmax = 30 +/- 15 fmol mg-1 protein). 3. Competition experiments were performed with ligands that are either non-selective for endothelin receptor subtypes. The rat cerebellar endothelin receptor displayed a high affinity for endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin-S6c (STX-6c) although the affinity for ET-3 was slightly higher than the affinity for ET-1 using both radioligands. The selective ETA antagonists, BQ123, BMS-182,874 and JKC-301 all displayed low affinities at the endothelin receptors. In contrast the selective ETB agonists, IRL1620 and [Ala1,3,11,15]ET-1 and the selective ETB antagonist, BQ-788 had moderate affinities at the endothelin receptor, in the low nanomolar range. The ETB agonist, BQ3020, had approximately 10 fold higher affinity than IRL1620 and [Ala1,3,11,15]ET-1 at the rat cerebellar endothelin receptors. The non-selective antagonists, Ro-46,2005, Ro-47,0203 and PD-142,893 displayed moderate affinities at the cerebellar receptor. 4. Since [125I]-BQ3020 recognises only a fraction of the [125I]-ET-1 binding sites, the majority of the endothelin receptors in the cerebellum cannot be classed as ETB. Although [125I]-PD151242 was able to detect ETA receptors in the rat cerebellar homogenates, the small population of ETA receptors (2% of the total endothelin population as measured with [125I]-ET-1) could not account for the non-ETB receptor population. We conclude that the rat brain cerebellar receptor has a profile similar to the ETB1 receptor as it has a high affinity for ET-1, ET-3, STX-6c and was moderately sensitive to PD-142,893. However, as the ETB ligands BQ-788, IRL1620 and [Ala1,3,11,15]ET-1 have only a moderate affinity for the rat cerebellar endothelin receptor and since ET-3 has a higher affinity as compared to ET-1, our findings suggest that the rat cerebellum contains predominately ETc receptors.     

Identification of selective, high affinity [125I]-angiotensin and [125I]-bradykinin binding sites in rat intestinal epithelia.

Specific [125I]-angiotensin II (AII) and [125I]-bradykinin (Bk) binding sites have been identified within epithelial membranes from rat jejunum and descending colon. These high affinity intestinal sites exhibited KD values of 0.64 +/- 0.16 nM for [125I]-AII and 0.69 +/- 0.13 nM for [125I]-Bk, which were similar to those for [125I]-AII (0.85 nM) and [125I]-Bk binding sites (1.03 nM) previously identified in renal cortex epithelia. Specific [125I]-AII binding capacity was only 19.77 +/- 2.74 fmol mg-1 in small intestine and 11.31 +/- 2.66 fmol mg-1 in descending colon epithelia while a larger population, 332.0 +/- 72.9 fmol-mg-1 of specific [125I]-Bk sites were identified in epithelial membranes from small intestine. Significant hydrolysis of both free [125I]-AII and [125I]-Bk was observed while membrane bound peptides remained relatively resistant to degradation. Whilst no corrections have been made to the observed values of KD and Bmax quoted above, one may assume that the calculated reductions in the free hormone concentration will result in a decrease of the KD value for both peptides. Loss of membrane bound peptide, particularly of [125I]-AII, may indicate that the calculated Bmax value is an underestimation. Despite the rapid degradation of unbound [125I]-AII and [125I]-Bk during incubations the kinetics of specific peptide binding were reversible and highly selective. The order of potency for specific [125I]-AII binding was [Sar1, Leu8]-AII greater than or equal to [Sar1, Thr8]-AII greater than or equal to AII greater than [Sar1, Ile8]-AII greater than or equal to [Des, Asp1, Ile8] AII greater than AIII. Specific [125I]-Bk binding was also highly selective, the order of potency being Phe8-Bk greater than or equal to Tyr8-Bk greater than or equal to Lys-Bk much greater than Des, Arg1-Bk. AII exhibited an IC50 of greater than 1mM for specific [125I]-Bk binding and likewise Phe8-Bk for specific [125I]-AII binding.     

Autoradiographic localisation of endothelin binding sites in kidney

[125I]Endothelin binding sites occurred in glomeruli of human, monkey, rat and dog kidney but not rabbit glomeruli. The renal medulla had more binding sites in all species, with the greatest densities of all in the rat and monkey renal papillas. Binding in rat kidney was inhibited by [Ala3,11]endothelin with lower potency than endothelin but not by unrelated peptides. These binding sites may represent the endothelin receptors and suggest actions for endothelin on renal function.     

Effect of minor groove binding drugs on mammalian topoisomerase I activity

Three minor groove binding drugs, distamycin A, bisbenzimide (Hoechst 33258) and 4',6-diamidino-2-phenylindole (DAPI), were examined for their abilities to modulate the activity of topoisomerase I purified from L1210 cells. At 0.5 and 1.0 microM, distamycin stimulated topoisomerase I relaxation of supercoiled DNA by 38 and 13%, respectively, while increasing the drug concentration above 2.0 microM resulted in inhibition. Inhibition was reversible. Complete relaxation could be achieved even in the presence of inhibitory concentrations of distamycin if the incubation time with topoisomerase I was increased from 7.5 to 120 min. The velocity of topoisomerase I mediated relaxation was reduced by 2 microM distamycin at DNA levels ranging from 350 to 2000 ng/reaction. Hoechst 33258 and DAPI inhibited topoisomerase I relaxation in a concentration-dependent manner. Hoechst 33258 and distamycin were equivalent in their abilities to inhibit topoisomerase I, whereas DAPI had a lesser effect (e.g. relaxation was reduced by 50% with 2.7 microM distamycin and 2.8 microM Hoechst 33258 compared to 5 microM DAPI). This study suggests that ligand binding in the minor groove can be a factor in the regulation of topoisomerase I activity.     

Autoradiographic localisation of [3H]2-BFI imidazoline I2 binding sites in mouse brain

Imidazoline I2 binding sites are heterogeneous in nature and have been observed in the brain of a number of species. Development of specific imidazoline I2 radioligands, such as [3H]2-BFI and [3H]BU224, that have a high affinity for the imidazoline I2 binding site, has enabled the central distribution of these sites to be mapped. Extensive studies have been conducted on the rat brain with a number of radioligands. However, to date a comprehensive analysis of imidazoline I2 ligand binding in mouse brain has not been completed. In the present work we describe levels of [3H]2-BFI specific binding found throughout the mouse brain. [3H]2-BFI (2 nM) showed discrete regional distribution which was readily displaced by saturating concentrations of the specific imidazoline I2 ligand BU224. The highest levels of [3H]2-BFI specific binding were found in the dorsal raphe, paraventricular thalamus and nucleus accumbens. Moderate levels were found throughout the lining of the aqueduct, lateral ventricle, lateral 4th ventricle, 4th ventricle, 3rd ventricle, but not the dorsal 3rd ventricle. Based on the loss of [3H]idazoxan binding in brain homogenates from monoamine oxidase-A and B (MAO-A and MAO-B) deficient mice it has been suggested that imidazoline I2 binding sites are predominantly on MAO. Consistent with this hypothesis the regional distribution of [3H]2-BFI shows some overlap with that previously reported for MAO. However, in the rat imidazoline I2 binding sites have been shown to be heterogeneous in nature and it is likely [3H]2-BFI is binding to multiple imidazoline I2 binding sites within mouse brain.     

Synthesis and binding of [125I2]philanthotoxin-343, [125I2]philanthotoxin-343-lysine, and [125I2]philanthotoxin-343-arginine to rat brain membranes.

125I2-iodinated philanthotoxin-343 (PhTX-343), [125I2]PhTX-343-arginine, and [125I2]PhTX-343-lysine were synthesized and evaluated as probes for glutamate receptors in rat brain synaptic membranes. It was found that these probes were not specific for the glutamate receptors but may be useful for investigating the polyamine binding site. Filtration assays with Whatman GF/B fiber glass filters were unsuitable because the iodinated PhTX-343 analogues exhibited high nonspecific binding to the filters, thus hindering detection of specific binding to membranes. When binding was measured by a centrifugal assay, [125I2]PhTX-343-lysine bound with low affinity (KD = 11.4 +/- 2 microM) to a large number of sites (37.2 +/- 9.1 nmol/mg of protein). The binding of [125I2]PhTX-343-lysine was sensitive only to the polyamines spermine and spermidine, which displaced [125I2]PhTX-343-lysine with Ki values of (3.77 +/- 1.4) x 10(-5) M and (7.51 +/- 0.77) x 10(-5) M, respectively. The binding was insensitive to glutamate receptor agonists and antagonists. Binding results with [125I2]PhTX-343-arginine were similar to those of [125I2]-PhTX-343-lysine. Considering the high number of toxin binding sites (10000-fold more than glutamate) in these membranes and the insensitivity of the binding to almost all drugs that bind to glutamate receptors, it is evident that most of the binding observed is not to glutamate receptors. On the other hand, PhTX analogues with photoaffinity labels may be useful in the isolation/purification of various glutamate and nicotinic acetylcholine receptors; they could also be useful in structural studies of receptors and their binding sites.     

Sedative potency and 2-[125I]iodomelatonin binding affinity of melatonin analogues

Melatonin (5-methoxyN-acetyltryptamine), the hormone synthesized and released from the pineal gland each night, has sedative and sleep-promoting effects in experimental animals and man. In the present study, the sedative effect of melatonin and a number of analogues was determined by examining their ability to extend the duration of the loss of righting reflex (sleeping time) in mice injected with pentobarbitone (50 mg/kg IV). All of the analogues tested produced a dose-related (5–20 mg/kg) potentiation of pentobarbitone sleeping time. In radioligand binding assays using 2-[¹²⁵I]iodomelatonin in chicken brain membranes, all of the analogues were competitive inhibitors. There was no correlation between their ability to inhibit 2-[¹²⁵I]iodomelatonin binding in chick and sedative potency in the mouse. Potentiation of pentobarbitone sleeping time by diazepam (1 mg/kg IP), but not melatonin (10 mg/kg IP), was blocked by pretreatment with the benzodiazepine antagonist, flumazenil (10 mg/kg IP). Similarly, an increase in pentobarbitone sleeping time produced by the aminoalkylindole cannabinoid receptor agonist, WIN 55212-2 (0.5 mg/kg IP), but not that produced by melatonin (10 mg/kg IP) was reduced by the cannabinoid receptor antagonist WIN 56098 (5 mg/kg IP). These studies confirm that melatonin has sedative activity and show that this action is shared by several structurally-related analogues but does not appear to be mediated by an interaction with benzodiazepine or cannabinoid receptors.     

Specific [125I]brain natriuretic peptide-26 binding sites in rat and pig kidneys

Specific binding sites for porcine brain natriuretic peptide-26 (BNP-26), a member of the atrial natriuretic peptide family (ANPs), were investigated in the kidney by using receptor autoradiographic and membrane binding techniques with [125I]BNP-26. The binding sites were discretely localized in rat and porcine kidney areas corresponding anatomically to the glomeruli and inner medulla. There were no differences between the localization of [125I]BNP-26 and [125I]alpha-rat ANP binding sites in the kidney. [125I]BNP-26 binding to solubilized membranes from isolated glomeruli of the rat kidney was saturable, and a single class of high-affinity sites was labeled with a KD of 372 pM. The radioligand bound to two sites in solubilized inner medullary membranes of the rat, a low-affinity site with a KD of 30 nM, and a high-affinity site with a KD of 33 pM. The rank order of potency to inhibit binding was BNP-26 = alpha-rat ANP-(1-28) greater than atriopeptin III (ANP-(103-126)) much greater than atriopeptin I (ANP-(103-123)) greater than des-Cys105,Cys121- ANP-(104-126). Thus, [125I]BNP-26 presumably recognizes ANP receptors in the kidney. The possibility that BNP-26 regulates, as a circulating hormone, kidney functions by binding to ANP receptors would have to be considered.     

Specific in vivo binding of [125I]-iodomelatonin to melatonin receptors in rat brain

The in vivo distribution of [125I]-2-iodomelatonin in rat brain was determined at different time intervals after intraarterial injection. After one hour, radioactivity in brain areas devoid of melatonin receptors had washed out to very low concentrations, but significant retention occurred in the medial basal hypothalamus (mbh) which contained the median eminence and in the anterior pituitary gland (ap), areas known to contain high concentrations of melatonin receptors. Coinjection of unlabelled melatonin reduced radioactivity concentrations in the ap and mbh by 44% and 75% respectively at one hour, whereas radiotracer concentrations in other regions remained unchanged. These results indicate the potential for the in vivo study of melatonin receptor concentration changes in human brain in disease states by means of single photon emission computed tomography.     

(-)[125I]pindolol binding to human peripheral lung beta-receptors

Beta-adrenergic receptors in human peripheral lung were characterized by biochemical and radioligand assays employing binding of the beta-antagonist (-)[125I]pindolol to plasma membrane preparations. The specific binding of (-)[125I]pindolol reached equilibrium by 45 min with an initial rate constant of 0.0282 min-1. Binding was reversible with a kinetic dissociation rate constant of 0.0146 min-1. The calculated kinetic Kd (dissociation constant) was 430 pM which agreed very well with the Kd of 394 pM obtained by Scatchard analyses of equilibrium binding data. Computer analyses of equilibrium binding experiments revealed a similar Kd of 336 +/- 24 pM. The binding capacities calculated by computer analyses (155 +/- 7 fmol/mg protein) and Scatchard analyses (113 fmol/mg protein) were also in close agreement. By all three methods (kinetic, Scatchard, and computer analyses), the data were most compatible with a single (-)[125I]pindolol binding site. Analyses of equilibrium binding data from ten different human lungs revealed values for the Kd ranging from 79 to 360 pM (mean, 136 pM), and for the receptor concentration ranging from 58 to 196 fmol/mg protein (mean, 118 fmol/mg protein). The displacement of (-)[125I]pindolol binding by various agents exhibited stereoselectivity and the expected rank order of potency predicted for interactions with beta-receptors. Isoproterenol induced a rapid and dose-related increase in cyclic AMP that was prevented by specific beta-antagonists. Approximately 70% of the beta-receptors were found to be of the beta 2-subtype by both radioligand binding and biochemical assays. Thus, (-)[125I]pindolol appears to be an excellent ligand for characterizing human lung beta-receptors since accurate and reproducible results can be obtained with this radioligand using limited tissue sample quantities.     

Similar distribution of [125I]sarafotoxin-6b and [125I]endothelin-1, -2, -3 binding sites in the human kidney

The distribution of binding sites for 125I-labelled endothelins (ET-1, ET-2, ET-3) and sarafotoxin-6b (SRTX-6b) was visualized in human kidneys using autoradiography. The glomeruli, the internal medulla and the renal blood vessels presented the highest levels of labelling, while the outer medulla exhibited intermediate and the cortex lower densities of sites. The relative enrichment of binding sites in different renal areas was similar for each peptide, suggesting the existence of a homogeneous population of endothelin/sarafotoxin receptors.     

Human platelet [125I]R-DOI binding sites. Characterization by in vitro autoradiography

We quantified binding sites for 2,5-dimethoxy-4-iodo-phenylisopropylamine (DOI), a 5-HT2 agonist and hallucinogen, in human platelets. We incubated sections from human platelet pellets with [125I]R-DOI with or without 1 mumol/L ketanserin, followed by autoradiography and computerized microdensitometry. We corrected the values of binding density by the protein content of each section with a densitometric protein assay. The present method revealed a single class of high affinity binding sites for [125I]R-DOI, with a Kd of 6.4 +/- 0.7 nmol/L and a Bmax of 100 +/- 10 fmol/mg protein. Kd and Bmax for [125I]R-DOI determined by the classical membrane binding assay, were 2.7 +/- 0.4 nmol/L and 100 +/- 10 fmol/mg protein, respectively. The present method is precise, very sensitive, and allows the characterization of [125I]R-DOI binding in sections obtained from as little as 3 ml of blood. Standardization is possible after correction by the protein content of each individual section.