Evidence for muscarinic cholinergic receptors in dog portal vein: binding of [3H]quinuclidinyl benzilate

Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 +/- 2.8 pM) with a Bmax of 110 +/- 14.7 fmol/mg protein. Scatchard and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 X 10(9) M-1 min-1 and 0.016 min-1, respectively, were calculated. The dissociation constant calculated from the equation KD = K-1/K+1 was 8.3 pM, such being in good agreement with the Scatchard estimate of KD (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, alpha-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at 10 microM. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.     

High affinity quinuclidinyl benzilate binding to rat parotid membranes requires muscarinic receptor. G protein interactions

The binding of the non-selective muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) to rat parotid membranes was characterized. Under equilibrium conditions, [3H]QNB bound to a homogenous population of muscarinic receptors (Kd, 118 +/- 19 pM; Bmax, 572 +/- 42 fmol/mg membrane protein, n = 12). The addition of G protein activators AlF4- or guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) + Mg2+ increased the Kd by 77 +/- 7% (n = 4, P less than 0.05) and 83 +/- 27% (n = 7, P less than 0.05), respectively, without a change in the Bmax or homogeneity of the binding site. GTP gamma S added without exogenous Mg2+ did not affect [3H]QNB binding. Thus, optimal QNB binding requires a muscarinic receptor/G protein interaction.     

Characterization of Muscarinic Cholinergic Receptors in the Crab Nervous System

The selective muscarinic antagonist L-[3H]-quinuclidinyl benzilate (L-[3H]QNB) binds reversibly and with high affinity (KD = 0.3 nM) to a single population (Bmax = 105 fmol/mg protein) of specific sites in nervous tissue of the crab Cancer magister. The binding site is stereoselective; (-)QNB is over 200 times more potent than (+)QNB as an inhibitor of specific L-[3H]QNB binding. The muscarinic antagonists scopolamine and atropine are over 10,000 times more potent inhibitors of L-[3H]QNB binding than the nicotinic antagonists decamethonium and d-tubocurarine. The muscarinic agonists oxotremorine, pilocarpine, arecoline, and carbachol also compete effectively for the L-[3H]QNB binding site. This pharmacological profile strongly suggests the presence of classical muscarinic receptors in the crab nervous system. These receptors are localized to nervous tissue containing cell bodies and neuropil, whereas specific L-[3H]QNB binding is low or absent in peripheral nerve, skeletal muscle, and artery.     

Characterization of muscarinic acetylcholine receptors in the avian salt gland

Electrolyte and fluid secretion by the avian salt gland is regulated by activation of muscarinic acetylcholine receptors (R). In this study, these receptors were characterized and quantitated in homogenates of salt gland from domestic ducks adapted to conditions of low (freshwater, FW) and high (saltwater, SW) salt stress using the cholinergic antagonist [3H]-quinuclidinyl benzilate (QNB). Specific binding of the antagonist to receptors in both FW- and SW-adapted glands reveals a single population of high affinity binding sites (KdFW = 40.1 +/- 3.0 pM; KdSW = 35.1 +/- 2.1 pM). Binding is saturable; RLmaxFW = 1.73 +/- 0.10 fmol/micrograms DNA; RLmaxSW = 4.16 +/- 0.31 fmol/micrograms DNA (where L is [3H]QNB and RL the high affinity complex). Calculated average cellular receptor populations of 5,800 sites/cell in FW-adapted glands and 14,100 sites/cell in SW-adapted glands demonstrate that upward regulation of acetylcholine receptors in the secretory epithelium follows chronic salt stress. The receptor exhibits typical pharmacological specificities for muscarinic cholinergic antagonists (QNB, atropine, scopolamine) and agonists (oxotremorine, methacholine, carbachol). In addition, the loop diuretic furosemide, which interferes with ion transport processes in the salt gland, competitively inhibits [3H]QNB binding. Preliminary studies of furosemide effects on [3H]QNB binding to rat exorbital lacrimal gland membranes showed a similar inhibition, although the diuretic had no effect on antagonist binding to rat brain or atrial receptors.     

Guanine Nucleotides Regulate 2-[125I]Iodomelatonin Binding Sites in Chick Retinal Pigment Epithelium but Not in Neuronal Retina

The characteristics of the binding sites labeled by the radioligand 2-[125I]iodomelatonin were compared in chicken neuronal retina and retinal pigment epithelium (RPE). Specific binding of 2-[125I]iodomelatonin in both sites was stable, saturable, reversible, and of high affinity. Scatchard analysis revealed an affinity constant (KD) of 446 +/- 55 pM and a total number of binding sites (Bmax) of 25.4 +/- 2.2 fmol/mg of protein for neuronal retina. For RPE the KD was 34.1 +/- 2.2 pM and the Bmax 59.5 +/- 5.2 fmol/mg of protein. Competition experiments with various melatonin analogues gave the following order of affinities: 2-iodomelatonin greater than 2-chloromelatonin greater than melatonin greater than 6-chloromelatonin greater than 6-hydroxymelatonin greater than N-acetylserotonin greater than 6-methoxyharmalan greater than 5-hydroxytryptamine. Linear regression of log Ki values from neuronal retina and RPE gave a highly significant correlation (r = 0.994, n = 8; p less than 0.001). GTP inhibited specific binding to RPE membranes in a concentration-dependent manner, but not in neuronal retinal membranes. The present results strongly suggest that a single type of melatonin receptor is found in neuronal retina and RPE, and that the site in RPE is coupled to a guanine nucleotide-binding regulatory protein (G protein), but that in neuronal retina is not.     

[3H]N-Methylscopolamine Binding Studies Reveal M2 and M3 Muscarinic Receptor Subtypes on Cerebellar Granule Cells in Primary Culture

Saturation experiments with the muscarinic antagonist [3H]N-methylscopolamine ([3H]NMS) indicated that cerebellar granule cells in primary culture possess a high density of muscarinic acetylcholine receptors (mAChRs): Bmax = 1.85 +/- 0.01 pmol/mg of protein at 10 days in culture; KD = 0.128 +/- 0.01 nM. The selective M1 antagonist pirenzepine displaced [3H]NMS binding with a low affinity (Ki = 273 +/- 13 nM), whereas the M2/M3 muscarinic antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide competed with [3H]NMS with Ki values in the nanomolar range, a result suggesting that some of the mAChRs on cerebellar granule cells belong to the M3 subtype. Methoctramine, which discriminates between M2 and M3 subtypes with high and low affinity, respectively, displayed a high and low affinity for [3H]NMS binding sites (Ki(H) = 31 +/- 5 nM; Ki(L) = 2,620 +/- 320 nM). These results provide the first demonstration that both M2 and M3 mAChR subtypes may be present on cultured cerebellar cells. In addition, complete death of neurons induced by N-methyl-D-aspartate (100 microM for 1 h) reduced by 85% the specific binding of [3H]NMS, a result indicating that most mAChRs were associated with neuronal components. Finally, the evolution of the density of mAChRs, labeled by [3H]NMS, correlated with the neuronal maturation during the in vitro development of these cells.     

Studies on the properties of muscarinic cholinergic receptor sites in bovine pineal gland

1. Using the tritiated muscarinic receptor antagonist, quinuclidinyl benzilate ([3H]QNB) as a ligand, muscarinic cholinergic receptors have been identified and characterized in the pineal glands of cow and swamp buffalo. 2. At 25 degrees C, the specific binding reached equilibrium within 60 min and remained constant for an additional two hours. Furthermore, the specific binding was saturable, reversible and tissue dependent in nature. 3. The kinetic analyses of muscarinic cholinergic receptor sites revealed KD values of 0.423 +/- 0.01 nM and 0.218 +/- 0.01 nM, and Bmax values of 69.75 +/- 20.91 fmol/mg protein and 74.19 +/- 32.73 fmol/mg protein for the cow's- and the swamp buffalo's pineal glands, respectively. 4. The presence of muscarinic cholinergic receptor sites originating from cholinergic innervation of the pineal gland is suggested.     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

Hormone effects on muscarinic cholinergic binding in bovine and rat sympathetic superior cervical ganglia

Muscarinic receptors were assessed by [3H]-quinuclidinyl benzilate (QNB) binding in 900 xg supernatants of bovine superior cervical ganglia (SCG). At 30 degrees C half maximal binding was reached within 3 min and equilibrium within 30 min. Scatchard analysis revealed a single population of binding sites with dissociation constant (Kd) = 0.15 +/- 0.01 nM and site concentration (Bmax) = 101 +/- 4 fmoles/mg prot. Binding was specific for muscarinic drugs. Incubation of bovine SCG with different hormones (10(-7)M) indicated that LH, TRH and testosterone depressed significantly Bmax, and that prolactin decreased both Kd and Bmax of [3H] -QNB binding. Several other hormones tested (TSH, GH, FSH, LHRH, angiotensin II, bradykinin, melatonin, estradiol, thyroxine and triiodothyronine) did not affect QNB binding. Hormone effects were not due to a direct interference with radioligand binding to membrane. The injection of LH to orchidectomized rats depressed Bmax of SCG QNB binding without changing the Kd. These results suggest that muscarinic cholinergic neurotransmission in SCG may be affected by hormones.     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.     

Autoradiographic Characterization and Localization of Quisqualate Binding Sites in Rat Brain Using the Antagonist [3H]6-Cyano-7-Nitroquinoxaline-2,3-Dione: Comparison with (R,S)-[3H]α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding Sites

Using quantitative autoradiography, we have investigated the binding sites for the potent competitive non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitro-quinoxaline-2,3-dione ([3H]-CNQX) in rat brain sections. [3H]CNQX binding was regionally distributed, with the highest levels of binding present in hippocampus in the stratum radiatum of CA1, stratum lucidum of CA3, and molecular layer of dentate gyrus. Scatchard analysis of [3H]CNQX binding in the cerebellar molecular layer revealed an apparent single binding site with a KD = 67 +/- 9.0 nM and Bmax = 3.56 +/- 0.34 pmol/mg protein. In displacement studies, quisqualate, L-glutamate, and kainate also appeared to bind to a single class of sites. However, (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) displacement of [3H]CNQX binding revealed two binding sites in the cerebellar molecular layer. Binding of [3H]AMPA to quisqualate receptors in the presence of potassium thiocyanate produced curvilinear Scatchard plots. The curves could be resolved into two binding sites with KD1 = 9.0 +/- 3.5 nM, Bmax = 0.15 +/- 0.05 pmol/mg protein, KD2 = 278 +/- 50 nM, and Bmax = 1.54 +/- 0.20 pmol/mg protein. The heterogeneous anatomical distribution of [3H]CNQX binding sites correlated to the binding of L-[3H]glutamate to quisqualate receptors and to sites labeled with [3H]AMPA. These results suggest that the non-NMDA glutamate receptor antagonist [3H]CNQX binds with equal affinity to two states of quisqualate receptors which have different affinities for the agonist [3H]AMPA.     

Iminodipropionitrile-Induced Dyskinesia in Mice: Striatal Calcium Channel Changes and Sensitivity to Calcium Channel Antagonists

Administration of 3,3'-iminodipropionitrile (IDPN) (1 g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN-induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200-110 binding sites (Bmax) in whole brains of IDPN-treated mice. Similarly, the K(+)-depolarization-dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 +/- 7 fmol/mg to 237 +/- 31 fmol/mg in control and treated groups, respectively) of PN 200-110 binding to the striatum without change of KD value (38 +/- 4.7 pM versus 33 +/- 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 +/- 10.1 pM to 45 +/- 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 +/- 51 fmol/mg versus 485 +/- 41 fmol/mg) of PN 200-110 binding to the cortex. IDPN did not alter omega-conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment as inhibited by L-type calcium channel antagonists and this may be associated with the observed increase in striatal L-type calcium channels.     

Separate Binding and Functional Sites for ω co-Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.     

Two polypeptides from Dendroaspis angusticeps venom selectively inhibit the binding of central muscarinic cholinergic receptor ligands.

Two new polypeptides were isolated and purified from the venom of the snake Dendroaspis angusticeps, which also contains other neuroactive peptides such as Dendrotoxins and Fasciculins. The amino acid composition of the peptides was determined and the first 10 amino acids from the MTX2 N-terminal fragment were sequenced. The so-called muscarinic toxins (MTX1 and MTX2) have been shown to inhibit the specific binding of [3H]QNB (0.15 nM), [3H]PZ (2.5 nM) and [3H]oxoM (2 nM) to bovine cerebral cortex membranes by 60, 88 and 82% respectively. In contrast, they caused only a 30% blockade of the [3H]QNB specific binding to similar membrane preparations from the brainstem. The Hill number for the [3H]PZ binding inhibition by the putative muscarinic toxin MTX2 was 0.95 suggesting homogeneity in the behaviour of the sites involved. The data from [3H]oxoM binding gave a Hill number of 0.83. The decreases in the specific binding involved increases in KD for the three different ligands (8-fold for [3H]QNB, 4-fold for [3H]PZ and 3.5-fold for [3H]oxoM) without significant changes in Bmax, except for a slight decrease in the [3H]oxoM binding sites (-19%); such results suggest that there may be a competitive inhibition between the MTXs and these ligands. The Ki for MTX2/[3H]PZ was 22.58 +/- 3.52 nM; for MTX2/[3H]oxoM, 144.9 +/- 21.07 nM and for MTX2/[3H]QNB, 134.98 +/- 18.35 nM. The labelling of MTX2 with 125I allowed direct demonstration of specific and saturable binding to bovine cerebral cortex synaptosomal membranes. In conclusion, the results reported in this study strongly support the hypotheses that the two polypeptides isolated from D. angusticeps venom selectively inhibit specific ligand binding to central muscarinic receptors, in a competitive manner at least for the antagonist [3H]PZ and that the MTX2 specifically binds to a central site that is suggested to be a muscarinic receptor of the M1 subtype.     

Decrease in rat cardiac beta 1- and beta 2-adrenoceptors by training and endurance exercise

The cardiac beta-adrenoceptor adaptation to physical activity was investigated in rats which were subjected to a six-week endurance swimming training (ET; n = 7) and a training of high intensity (MT; n = 7). In addition, the effect of a single bout of endurance exercise without preceding training (EE; n = 7) was evaluated. These groups were compared with a sedentary control group (C; n = 9). Beta-adrenergic receptors in rat myocardial membranes were labelled using the high affinity antagonist radioligand (-)125iodocyanopindolol (ICYP). Computer modelling techniques provided estimates of the maximal binding capacity (Bmax) and the dissociation constants (KD). Tissue was constantly kept at temperatures of less than or equal to 4 degrees C and incubated at 4 degrees C for 18 h in buffer containing 100 microM GTP so as to prevent masking of beta-adrenoceptors by endogenous norepinephrine. In comparison with the C group (Bmax = 43.2 +/- 1.6 fmol/mg protein, KD = 11.7 +/- 1.5 pM) computerized coanalyses of saturation binding data of ET, MT, and EE revealed a 13.0%, 25.5%, and 16.6% decrease in Bmax (P less than 0.01), respectively, without significantly differing KD values (10.6 pM, 9.0 pM, 10.5 pM, respectively). We provide the first evidence that acute exercise lowers the sarcolemmal beta-adrenoceptor number in the rat heart. In the competition radioligand binding, CGP20712A and ICI118.551 were employed as subtype-selective antagonists of beta 1- and beta 2-adrenoceptors, respectively, to determine the relative proportions of the receptor subtypes. The ratio of beta 1-/beta 2-adrenoceptors in C was 67.5:32.5 and no statistically significant variation occurred in animals subjected to physical activity. On the basis of published data we assume that acute exercise induces a sequestration of beta-adrenoceptors from the cell surface to some intracellular compartment, whereas the molecular basis of the chronic beta-adrenoceptor down-regulation may involve a training-induced reduction in receptor synthesis. Our findings on cardiac beta-adrenoceptor adaptation to physical activity may represent one of the mechanisms underlying the relative bradycardia in trained subjects.     

3-[(3-Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate-Solubilized Binding Sites for 2-[125I]Iodomelatonin in Chick Brain Retain Sensitivity to Guanine Nucleotides

Binding of 2-[125I]iodomelatonin to 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized sites from chick forebrain was rapid. reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2-[125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 +/- 22 (n = 4) and 302 +/- 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 +/- 2.0 and 49.5 +/- 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than prazosin greater than N-acetylserotonin much greater than serotonin greater than metergoline greater than ketanserin greater than propranolol greater than phentolamine greater than cyproheptadine. Guanyl nucleotides inhibited binding of 2-[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high-affinity to a low-affinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane-bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.     

Binding of 125I-labelled human chorionic gonadotropin to cell membrane receptors in rabbit ovarian slices

The binding of 125I-labelled human chorionic gonadotropin (HCG) was studied using thick slices (300 micron) of rabbit ovarian tissue. Binding was saturable, reversible, stereospecific, and of high affinity. The amount of binding was proportional to the number of slices used and could be destroyed by boiling. Ovarian slices from eight individual rabbits were found to have two binding sites for 125I-labelled HCG with KD values of 272 +/- 64 and 1263 +/- 274 pM and Bmax values of 25.7 +/- 5.3 and 94.1 +/- 18.8 fmol/mg protein, respectively. In a comparative study the KD and Bmax values were 351 +/- 151 pM and 25.3 +/- 11.1 fmol/mg protein with slices from one ovary and 134 +/- 24 pM and 109 +/- 32 fmol/mg protein with membranes from the contralateral ovary. These data suggest that the binding of HCG can be determined in live tissue.     

Muscarinic acetylcholine receptors in the brain of the zebrafish (Danio rerio) measured by radioligand binding techniques

Muscarinic acetylcholine receptors (mAChRs) play a role in learning, memory and behavior in vertebrate animals. We measured the muscarinic cholinergic receptor levels in extracts from zebrafish (Danio rerio) brain by radioligand binding techniques. Saturation binding experiments with the radioligand [3H]-quinuclidinyl benzilate (QNB) were used to determine receptor number and relative affinity for several agonists and antagonists. Affinity at zebrafish brain receptors was relatively high with a K(d) of 40 +/- 5 pM. The number of receptors, represented by Bmax, was 63 +/- 16 fmol/mg protein. Oxotremorine and carbachol, agonists at muscarinic acetylcholine receptors, bound with displacement curves indicating multiple binding sites. In addition, oxotremorine bound with a higher affinity than did carbachol. The antagonist potency profile at zebrafish receptors in brain was determined to be atropine>pirenzipine>p-fluoro-hexahydro-sila-difenidol>otenzepad. The results obtained with zebrafish brain compare favorably to those found in insect, fish and mammalian species. Taken together, the binding results and favorable comparisons to mammalian systems indicate that zebrafish may provide a useful model organism for evaluating the role of cholinergic systems in learning, memory and behavior.     

Muscarinic Cholinergic Receptors in Human Foetal Brain: Characterization and Ontogeny of [3H]Quinuclidinyl Benzilate Binding Sites in Frontal Cortex

Twenty-two frontal cortices from normal human foetal brains of gestational ages ranging from 16 to 40 weeks and five postnatal brains ranging from 5 to 50 years were analysed for the ontogeny of muscarinic receptors using [3H]quinuclidinyl benzilate (QNB) as the ligand. QNB binding sites were shown to be stable up to 4 1/2 months of storage at -70 degrees C. QNB binding was characterized in frontal cortices of 28-week-old foetal brains as muscarinic receptors by the following criteria: (1) it was localised mainly in particulate fraction; (2) binding was saturable at a concentration of 1.5 nM; (3) the cholinergic antagonists atropine and scopolamine competed for the binding, with IC50 values of 1 and 0.8 nM, respectively. The agonists oxotremorine, carbachol, and pilocarpine gave IC50 values of 1, 15 and 18 microM, respectively. Nicotinic receptor ligands and noncholinergic drugs could not compete for the binding. Bimolecular association and dissociation rate constants for the reversible binding are 6.23 X 10(8) M-1 X min-1 and 2.0 X 10(-2) X min-1, respectively. The equilibrium dissociation constant is 33 pM. The KD obtained by saturation binding data is 103 pM. Ontogeny of muscarinic receptors showed three distinct phases: In phase I, they appear between 16 and 18 weeks [average concentration 109 fmol/mg protein of total particulate fraction (TPF)] and slowly increase up to 20 weeks (average concentration 147 fmol/mg protein TPF). Phase II is a lag period between 20 and 24 weeks at which time receptor concentration does not change perceptibly (average concentration (67 fmol/mg protein TPF).(ABSTRACT TRUNCATED AT 250 WORDS)