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.     

Characterization of propranolol-resistant (-)-[125I]-cyanopindolol binding sites in rat soleus muscle.

1. The characteristics of a propranolol-resistant (-)-[125I]-cyanopindolol (CYP) binding site in rat soleus muscle were determined. 2. Saturation studies performed on homogenates of rat soleus muscle showed two phases of (-)-[125I]-CYP binding, a high affinity site (KD1 30.5 +/- 16.3 pM, Bmax 9.4 +/- 1.38 fmol mg-1 protein) and a lower affinity site (KD2 522.5 +/- 29.1 pM, Bmax 62.19 +/- 11.76 fmol mg-1 protein, n = 4). 3. In rat soleus muscle homogenates labelled with (-)-[125I]-CYP (500 pM), (-)-propranolol competition curves were biphasic with pKD values of 8.30 +/- 0.19, and 5.33 +/- 0.08, n = 7. 4. Competition between (-)-[125I]-CYP (500 pM) and (+/-)-tertatolol, (+/-)-nadolol, (+/-)-alprenolol, (+/-)-CYP, and (-) and (+)-pindolol showed that these compounds competed for binding at the propranolol-resistant site with affinities lower than those displayed at typical beta-adrenoceptors. The atypical beta-adrenoceptor agonists BRL 37344, SR58611A and ICI D7114 and the partial agonist (+/-)-CGP 12177 also competed for (-)-[125I]-CYP binding. 5. Stereoselectivity was demonstrated for the stereoisomers of alprenolol and tertalolol. The (-)-isomers of alprenolol and tertalolol had higher affinity than their corresponding (+)-isomers (3.1 and 2.6 fold respectively). These low stereoselectivity values are a characteristic of atypical beta-adrenoceptors. 6. The beta-adrenoceptor agonists, (-)-adrenaline, (-)-isoprenaline and (-)-noradrenaline, all showed lower affinity than the atypical beta-adrenoceptor agonists and competition curves appeared biphasic in nature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the bradykinin receptor in the human nasal airway using the binding of [125I]-Hoe 140.

1. The aim of this study was to characterize the kinin receptor in the human nasal airway using [125I]-Hoe 140 binding to a membrane preparation from human nasal turbinates and to compare Ki values from binding displacement by antagonists with the functional effects of these drugs in vivo. We also investigated the effect of Hoe 140 ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin), on bradykinin release into the nasal airway. 2. In a membrane preparation from human nasal turbinates removed during surgery, [125I]-Hoe 140 labelled a single, saturable binding site. The equilibrium dissociation constant (at 20 degrees C) for [125I]-Hoe 140 binding to the receptor was 0.46 +/- 0.08 nM. The Bmax was 0.136 +/- 0.003 pmol mg-1 protein and the Hill coefficient was 1.01 +/- 0.07. 3. The association rate constant for [125I]-Hoe 140 binding to the receptor was 0.20 +/- 0.06 nM-1 min-1 and the dissociation rate constant was 0.14 +/- 0.01 min-1. These values were determined at 4 degrees C. The equilibrium dissociation constant calculated from these rate constants was 0.70 nM. 4. Bradykinin and the B2 receptor antagonists, NPC 567, NPC 17731, NPC 17761, [1-adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, WIN 64338 and Hoe 140 displaced [125I]-Hoe 140 binding: the Ki values from binding displacement are consistent with values expected from a B2 receptor. The B1 agonist, [des-Arg9]-bradykinin and the B1 antagonist, [des-Arg9]-Hoe 140 failed to displace [125I]-Hoe 140 binding at concentrations up to 1 microM. 5. The bradykinin antagonist, Hoe 140, 10 to 200 micrograms, given by intranasal aerosol, produced a dose-related inhibition of the reduction in minimal nasal cross-sectional area (Amin) induced by bradykinin in normal subjects and by house dust mite antigen in subjects with allergic rhinitis to house dust mite. Hoe 140, 10 to 200 micrograms, also caused a dose-related inhibition of the release of albumin into the nasal cavity following challenge with bradykinin. 6. [1-Adamantane acetyl-D-Arg0, Hyp3, Thi5,8, D-Phe7]-bradykinin, 30 to 200 micrograms, caused a dose-related inhibition of the reduction in Amin and the release of albumin into the nasal cavity induced by bradykinin. NPC 567 ([D-Arg0, Hyp3, D-Phe7]-bradykinin) failed to inhibit the reduction in Amin or the release of albumin into the nasal cavity at a dose of 10 mg. 7. Challenge of allergic subjects with house dust mite antigen caused a significant elevation of the bradykinin concentration in nasal lavage fluid and a reduction in Amin. Hoe 140, 100 micrograms, prevented the antigen-induced reduction in Amin and also abolished the antigen-induced increase of bradykinin in nasal lavage fluid. 8. We conclude that there is a B2 bradykinin receptor in the human nasal airway which mediates nasal blockage and plasma extravasation induced by either bradykinin or antigen challenge. It is possible that Hoe 140 inhibits kallikrein in the human nasal airway as well as blocking the B2 receptor.     

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.     

Characterization of two new ETB selective radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 in human heart.

Two new endothelin receptor radioligands, [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1, were characterized in tissue sections of human right atrium and left ventricle. Both radioligands had high affinity ([125I]-BQ3020 right atrium: KD = 0.145 +/- 0.037 nM, left ventricle: KD = 0.107 +/- 0.004 nM; [125I]-[Ala1,3,11,15]ET-1 right atrium: KD = 0.239 +/- 0.036 nM, left ventricle: KD = 0.199 +/- 0.027 nM). Competition binding experiments were performed in the left ventricle. The selective ETA receptor compound BQ123 competed with low affinity against [125I]-BQ3020 (KD = 28.7 +/- 2.7 microM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 28.5 +/- 4.2 microM). The selective ETB receptor compound BQ3020 competed with high affinity against [125I]-BQ3020 (KD = 40.8 +/- 6.6 pM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.276 +/- 0.099 nM). Another selective ETB receptor compound, [Ala1,3,11,15]ET-1 also competed with high affinity against [125I]-BQ3020 (KD = 0.663 +/- 0.120 nM) and [125I]-[Ala1,3,11,15]ET-1 (KD = 0.643 +/- 0.124 nM). These results indicate that [125I]-BQ3020 and [125I]-[Ala1,3,11,15]ET-1 are selective ETB receptor radioligands. [Ala1,3,11,15]ET-1 competed with the non-selective radioligand [125I]-ET-1 in left ventricle and revealed the presence of ETA and ETB receptors in the proportions of 76:24% respectively in the human left ventricle.     

GTP modulates [125I]iodomelatonin binding to a picomolar-affinity site in the Syrian hamster hypothalamus

Saturation binding experiments conducted with [125I]iodomelatonin at 0-4 degrees C in the Syrian hamster hypothalamus, revealed a single nanomolar-affinity site which was not affected by GTP. In contrast, incubation at 30 degrees C revealed two distinct binding sites with picomolar and nanomolar affinities, respectively. GTP caused a significant decrease in the affinity of only the picomolar site but did not alter its density; control: Kd = 43 +/- 6 pM, Bmax = 1.7 +/- 0.3 fmol/mg protein; GTP (1 mM): Kd = 250 +/- 52, Bmax = 3.9 +/- 2.6 fmol/mg protein. The foregoing indicates that the affinity of the putative melatonin receptor in the hamster hypothalamus is modulated by a regulatory G protein.     

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.     

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.     

Characterization of binding sites for [125I]R(+)trans-7-OH-PIPAT in rat brain

Binding characteristics of a novel radioiodinated ligand, [¹²⁵I]R(+)trans-7-hydroxy-2-(N-n-propylN-3-iodo-2-propenyl) aminotetralin (¹²⁵I]R(+)trans-7-OH-PIPAT), were evaluated using homogenate binding and autoradiographic techniques in rat brain. [¹²⁵I]R(+)trans-7-OH-PIPAT bound to sites (dopamine receptors) in homogenates of rat basal forebrain (including caudate putamen, nucleus accumbens and olfactory tubercle) with a high affinity (K_d = 0.42 nM). A majority (70%) of the sites labeled by [¹²⁵I]R(+)trans-7-OH-PIPAT in basal forebrain were GTP-sensitive. In rat hippocampal homogenates, specific and saturable binding of [¹²⁵I]R(+)trans-7-OH-PIPAT to 5-HT_1A receptors, with a K_d value of 1.4 nM and a B_max value of 210 fmol/mg protein, was observed. Binding of [¹²⁵I]R(+)trans-7-OH-PIPAT to sigma sites was also demonstrated in rat cerebellar homogenates. In the presence of GTP (to inhibit binding to D 2 and 5-HT_1A receptors) and DTG (to inhibit binding to sigma sites), dopamine D3 receptors could be selectively labeled with [¹²⁵I]R(+)trans-7-OH-PIPAT. [¹²⁵I]R(+)trans-7-OH-PIPAT offers several unique advantages, including high specific activity and high affinity binding, which make it an excellent probe for the investigation and characterization of the distribution of dopamine D 3 receptors.Abbreviations 7-OH-DPAT 7-hydroxy-2-N,N-(di-n-propyl)aminotetralin - trans-7-OH-PIPAT traps-7-hydroxy-2-(N-n-propyl-N-3-iodo-2propenyl)aminotetralin - 8-OH-DPAT 8-hydroxy-2-N,N-(di-n-propyl)aminotetralin - 8-OH-PIPAT trans-8-hydroxy-2-(N-n-propyl-N-3-iodo-2-propenyl)aminotetralin - NCQ298 S(–)-3-iodo-N-[(1-ethyl-2-pyrrolidinyl)]methyl-2-hydroxy-5,6-dimethoxybenzamide - DTG 1,3-diortho-tolyl-guanidine - Sf9 cells Spodoptera frugiperda insect cells - CHO cells Chinese hamster ovary cells - GTP guanosine 5-triphosphate - WB4101 2-(2,6-dimeethoxyphenoxyethyl)aminomethyl-1,4benzodioxane - R(+)3PPP R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine - (+)MK-801 (+)5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine     

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.     

Characterization of rat liver beta-adrenoceptors during perinatal development as determined by [125I]-iodopindolol radioligand binding assays.

1. The subtype specificity of beta-adrenoceptors in foetal (20 days post coitum) rat liver membrane preparations has been determined by use of [125I]-iodopindolol binding assays and the characteristics of radioligand binding have been resolved. 2. The kinetics of radioligand association and dissociation (in the presence of 5 x 10(-4) M isoprenaline) showed an association rate constant of 1.5 x 10(7) M-1 S-1 and dissociation rate constant of 9.1 x 10(-4) S-1, corresponding to a dissociation constant for [125I]-iodopindolol of 60.7 pM. A similar dissociation constant (75 pM) was determined by saturation binding assays. 3. The rank order of potency for displacement of [125I]-iodopindolol binding was consistent with binding to a predominantly beta 2-adrenoceptor population (i.e. ICI 118551 greater than isoprenaline greater than adrenaline greater than noradrenaline greater than atenolol). Computer analysis of displacement curves in the presence of a beta 1-subtype selective agent (atenolol) or a beta 2-subtype selective agent (ICI 118551) revealed the presence of beta 2- and beta 1-adrenoceptor subtypes in a ratio of about 80:20%. 4. Saturation binding assays by use of [125I]-iodopindolol were carried out at different perinatal ages to determine total beta-adrenoceptor concentrations and beta 2-subtype (in the presence of 5 x 10(-7) M atenolol) adrenoceptor concentrations. Competition binding assays with atenolol confirmed that at all ages apparent beta 2-adrenoceptor binding accounted for 84-95% of the total beta-adrenoceptor binding. The total beta- and beta 2-adrenoceptor binding capacity increased by 2.3 fold from 20 days post coitum to birth, and then decreased postnatally at 1 and 2 days post partum. The dissociation constant for [125I]-iodopindolol binding did not show any change with age. 5. The change in beta 2-adrenoceptor concentration with age is discussed in relation to the changing beta-adrenoceptor-mediated responsiveness of glucose production by rat liver during perinatal development.     

Characterization of the snake venom ligand [125I]-DNP binding to natriuretic peptide receptor-A in human artery and potent DNP mediated vasodilatation

BACKGROUND AND PURPOSE: The natriuretic peptides, ANP and BNP, modulate vascular smooth muscle tone in human conduit arteries. Surprisingly, the natriuretic peptide receptor-A (NPR-A) has not been visualized using radioligand binding in these vessels. A new member of this peptide family, Dendroaspis natriuretic peptide (DNP) identified from snake venom, has been proposed to be present in human plasma and endothelial cells. Also, recently a novel radioligand, [(125)I]-DNP, has been characterized as selective for NPR-A in human heart. EXPERIMENTAL APPROACH: Our aims were to investigate expression and function of NPR-A receptors in human mammary artery using [(125)I]-DNP to quantify receptor density, immunocytochemistry to delineate the cellular distribution of the receptor and in vitro pharmacology to compare DNP induced vasodilatation to that of ANP. KEY RESULTS: Saturable, sub-nanomolar affinity [(125)I]-DNP binding was detected to smooth muscle of mammary artery, with receptor density of approximately 2 fmol mg(-1) protein, comparable to that of other vasoactive peptides. NPR-A immunoreactivity was localised to vascular smooth muscle cells and this was confirmed with fluorescence dual labelling. NPR-A expression was not detected in the endothelium. Like ANP, DNP fully reversed the constrictor response to ET-1 in endothelium intact or denuded mammary artery, with comparable nanomolar potencies. CONCLUSIONS AND IMPLICATIONS: This is the first characterization of NPR-A in human mammary artery using [(125)I]-DNP and we provide evidence for the presence of receptor protein on vascular smooth muscle cells, but not endothelial cells. This implies that the observed vasodilatation is predominantly mediated via direct activation of smooth muscle NPR-A.     

Characterization of muscarinic receptors mediating contractions of circular and longitudinal muscle of human isolated colon.

1. The effects of seven muscarinic receptor antagonists were used to characterize the receptors which mediate carbachol-evoked contractions of intertaenial circular and taenial longitudinal muscle in human isolated colon. The effects of these antagonists were studied upon colon contractions induced by cumulatively added carbachol which had mean EC50 values of 11.7 +/- 2.3 microM (n = 8) and 12.6 +/- 2.3 microM (n = 8) respectively upon circular and longitudinal smooth muscle. 2. All antagonists displaced concentration-response curves to carbachol to the right in a parallel manner. The maximum concentration of each antagonist added (30 nM-10 microM) did not significantly suppress the maximum response. 3. In circular muscle, the M3 muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), hexahydrosiladiphenidol (HHSiD) and para-fluoro-hexahydrosiladiphenidol (p-F-HHSiD) inhibited responses with pA2 values of 9.41 +/- 0.23, 7.17 +/- 0.07, 6.94 +/- 0.18 respectively. The M2 muscarinic receptor antagonist, AF-DX 116, the M2/M4 muscarinic receptor antagonist, himbacine, and the M1 muscarinic receptor antagonist, pirenzepine, yielded pA2 values of 7.36 +/- 0.43, 7.47 +/- 0.14 and 7.23 +/- 0.48 respectively. The non-selective antagonist, atropine, had a pA2 of 8.72 +/- 0.28. 4. In longitudinal muscle 4-DAMP, HHSiD, p-F-HHSiD, AF-DX 116, himbacine and pirenzepine gave pA2 values of 9.09 +/- 0.16, 7.45 +/- 0.43, 7.44 +/- 0.21, 6.44 +/- 0.1, 7.54 +/- 0.40, 6.87 +/- 0.38 respectively. Atropine yielded a pA2 value of 8.60 +/- 0.08.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of [125I]-angiotensin to rat renal epithelial cell membranes.

1 Specific high affinity binding sites for [125I]-angiotensin II have been identified in crude basolateral and brush border membranes from rat cortex. 2 A central high affinity site, KD 0.62 nM; Bmax 299 fmol/mg was identified as part of a complex multicomponent binding system. 3 This high affinity site was saturable and exhibited specificity for angiotensin II analogues and closely related peptides but not for bradykinin, substance P or peptide fragments of angiotensin II. 4 Specific [125I]-angiotensin II binding was partially dependent on NaCl. Absence of NaCl resulted in a decrease in Bmax, had no effect on the rate of association but increased the rate of dissociation of [125I]-angiotensin from its binding site.     

Localization and characterization of two propranolol resistant (-) [125I]cyanopindolol binding sites in rat skeletal muscle.

Autoradiographic studies were performed in sections of rat gastrocnemius, plantaris and soleus muscle bundles with (-)-[125I]cyanopindolol (59-69 pM) in the presence of (-)-propranolol (1 microM) to block beta 1- and beta 2-adrenoceptors. Two distinct populations of binding sites remained, one evenly distributed over the muscle bundles and the other localized in discrete patches. Evenly distributed binding was highest in the soleus muscle and inhibited by (+/-)-, (-)- and (+)-alprenolol (20 microM), tertatolol (1 microM), BRL 37344 (2-20 microM), (-)-isoprenaline (100 microM), phentolamine (10 microM) and haloperidol (250 microM) but not ICI 118,551 (70 nM), CGP 20712A (100 nM), (+)-isoprenaline (100 microM), pindolol (2 microM), cimaterol (100 microM) or serotonin (10 microM). Stereoselectivity for the optical isomers of alprenolol was displayed in the soleus muscle only. Highly localized binding was inhibited by serotonin (10 microM), (-)- and (+)-isoprenaline (100 microM) and phentolamine (10 microM).     

Characterization and modulation of [125I]iberiotoxin-D19Y/Y36F binding in the guinea-pig urinary bladder

The radioligand binding characteristics of the Ca(2+)-activated K(+) channel ligand [125I]iberiotoxin-D19Y/Y36F were examined in guinea-pig urinary bladder membranes. Saturation analysis revealed a single class of high affinity binding sites in the bladder with a K(D) value of 45.6 pM and a B(max) value of 112 fmol/mg protein. Specific binding was displaced by unlabeled iberiotoxin and penitrem A, but not by blockers of other classes of K(+) channels including alpha-dendrotoxin, margatoxin and apamin. The indole alkaloids, paxilline and verruculogen, significantly increased binding by 4.5- and 4.3-fold, respectively. Tetraacetic acid derivatives such as ethylenediamine tetraacetic acid and ethyleneglycoltetraacetic acid enhanced specific [125I]iberiotoxin-D19Y/Y36F binding about 2.5-fold, which was not attributable to calcium chelation. This increase was due to a significant change in ligand binding affinity (K(D)=6.3 pM), but not due to a change in the B(max), indicating that these compounds may enhance toxin binding via allosteric interactions. Collectively, these results demonstrate that the binding sites for [125I]iberiotoxin-D19Y/Y36F present in the urinary bladder shows a pharmacological profile typical of maxi-K(+) channels and can be modulated, not only by previously known indole alkaloids, but also by tetraacetic acid analogs.