Radiolabelled Antagonist Research Articles

A live cell NanoBRET binding assay allows the study of ligand-binding kinetics to the adenosine A3 receptor

There is a growing interest in understanding the binding kinetics of compounds that bind to G protein-coupled receptors prior to progressing a lead compound into clinical trials. The widely expressed adenosine A3 receptor (A3AR) has been implicated in a range of diseases including immune conditions, and compounds that aim to selectively target this receptor are currently under development for arthritis. Kinetic studies at the A3AR have been performed using a radiolabelled antagonist, but due to the kinetics of this probe, they have been carried out at 10 °C in membrane preparations. In this study, we have developed a live cell NanoBRET ligand binding assay using fluorescent A3AR antagonists to measure kinetic parameters of labelled and unlabelled compounds at the A3AR at physiological temperatures. The kinetic profiles of four fluorescent antagonists were determined in kinetic association assays, and it was found that XAC-ser-tyr-X-BY630 had the longest residence time (RT = 288 ± 62 min) at the A3AR. The association and dissociation rate constants of three antagonists PSB-11, compound 5, and LUF7565 were also determined using two fluorescent ligands (XAC-ser-tyr-X-BY630 or AV039, RT = 6.8 ± 0.8 min) as the labelled probe and compared to those obtained using a radiolabelled antagonist ([3H]PSB-11, RT = 44.6 ± 3.9 min). There was close agreement in the kinetic parameters measured with AV039 and [3H]PSB-11 but significant differences to those obtained using XAC-S-ser-S-tyr-X-BY630. These data indicate that selecting a probe with the appropriate kinetics is important to accurately determine the kinetics of unlabelled ligands with markedly different kinetic profiles.

Comprehensive evaluation of a somatostatin-based radiolabelled antagonist for diagnostic imaging and radionuclide therapy

Targeting of tumours positive for somatostatin receptors (sst) with radiolabelled peptides is of interest for tumour localization, staging, therapy follow-up and targeted radionuclide therapy. The peptides used clinically are exclusively agonists, but recently we have shown that the radiolabelled somatostatin-based antagonist (111)In-DOTA-sst2-ANT may be preferable to agonists. However, a comprehensive study of this radiolabelled antagonist to determine its significance was lacking. The present report describes the evaluation of this novel antagonist labelled with (111)In and (177)Lu in three different tumour models. Radiopeptide binding, internalization and dissociation studies were performed using cells expressing HEK293-rsst(2). Biodistribution studies were performed in HEK293-rsst(2), HEK293-hsst(2) and HEK293-rsst(3) xenografted mice. Saturation binding analysis confirmed earlier IC(50) data for (111/nat)In-DOTA-sst2-ANT and showed similar affinity of (177/nat)Lu-DOTA-sst2-ANT for the sst(2). Only low internalization was found in cell culture (6.68 ± 0.06 % at 4 h), which was not unexpected for an antagonist, and this could be further reduced by the addition of sucrose. No internalization was observed in HEK293 cells not expressing sst. Both results indicate that the internalization was specific. (111)In-DOTA-sst2-ANT and (177)Lu-DOTA-sst2-ANT were shown to target tumour xenografts expressing the rat and the human sst(2) receptor with no differences in their uptake or pharmacokinetics. The uptake in rsst(2) and hsst(2) was high (about 30 %IA/g 4 h after injection) and surprisingly long-lasting (about 20-23 %IA/g 24 h after injection). Kidney uptake was blocked by approximately 50 % by lysine or Gelofusine. These results indicate that radiolabelled somatostatin-based antagonists may be superior to corresponding agonists. The long tumour retention time of (177)Lu-DOTA-sst2-ANT indicates that this new class of compounds is of relevance not only in diagnostic imaging but also in targeted radionuclide therapy of sst-positive tumours.

Ser352 and Ser354 in the carboxyl terminus of the human P2Y(1) receptor are required for agonist-promoted phosphorylation and internalization in MDCK cells.

The P2Y(1) receptor promotes chloride secretion in epithelial cells, a process critical for regulation of extracellular ion and fluid levels. Here we have examined the role of phosphorylation in agonist-induced internalization of P2Y(1) receptors. A high-affinity radiolabelled antagonist, MRS2500, was used to quantify cell surface-binding sites of P2Y(1) receptors in Madin-Darby canine kidney (MDCK) epithelial cells, following exposure to agonists. The regions in the carboxyl terminus involved in both agonist-induced internalization of the receptor and its phosphorylation were identified by mutational analysis. Endogenous and stably expressed recombinant P2Y(1) receptors rapidly internalized with similar time courses in response to agonist in MDCK cells, ensuring that the levels of recombinant receptor achieved by retroviral infection did not adversely affect function of the internalization machinery. Four protein kinase C inhibitors of varying specificity did not affect internalization of recombinant receptors. Agonist-promoted internalization of a series of truncated P2Y(1) receptors identified a region between residues 349 and 359 in the carboxyl terminus as critical for regulation. Two amino acids within this region, Ser352 and Ser354, were shown to be both necessary and sufficient for agonist-promoted receptor phosphorylation and internalization. Our results firmly establish Ser352 and Ser354 in the carboxyl terminus of P2Y(1) receptors as critical residues for agonist-induced receptor internalization in MDCK cells. As the mechanism mediating this regulation requires phosphorylation of these key residues, the relevant receptor-regulated protein kinase can now be identified.

Specific labelling of serotonin 5-HT 1B receptors in rat frontal cortex with the novel, phenylpiperazine derivative, [ 3H]GR125,743 : A pharmacological characterization

Although several tritiated agonists have been used for radiolabelling serotonin (5-hydroxytryptamine, 5-HT) 1B receptors in rats, data with a selective, radiolabelled antagonist have not been presented. Inasmuch as [ 3H]GR125,743 specifically labels cloned, human and native guinea pig 5-HT 1B receptors and has been employed for characterization of cerebral 5-HT 1B receptor in the latter species [Eur. J. Pharmacol. 327 (1997) 247.], the present study evaluated its utility for characterization of native, cerebral 5-HT 1B sites in the rat. In homogenates of frontal cortex, [ 3H]GR125,743 (0.8 nM) showed rapid association ( t 1/2=3.4 min), >90% specific binding and high affinity ( K d=0.6 nM) for a homogeneous population of receptors with a density ( B max) of 160 fmol/mg protein. In competition binding studies, affinities were determined for 15 chemically diverse 5-HT 1B agonists, including 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1 H-indole-3-yl]ethylamine (L694,247; p K i, 10.4), 5-carboxamidotryptamine (5-CT; 9.7), 3-[3-(2-dimethylamino-ethyl)-1 H-indol-6-yl]- N-(4-methoxybenzyl)acrylamide (GR46,611; 9.6), 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1 H-indole (RU24,969; 9.5), dihydroergotamine (DHE; 8.6), 5- H-pyrrolo[3,2- b]pyridin-5-one,1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl (CP93,129; 8.4), anpirtoline (7.9), sumatriptan (7.4), 1-[2-(3-fluorophenyl)ethyl]-4-[3-[5-(1,2,4-triazol-4-yl)-1 H-indol-3-yl]propyl]piperazine (L775,606; 6.4) and (−)-1( S)-[2-[4-(4-methoxyphenyl)piperazin-1-yl]ethyl]- N-methyl-3,4-dihydro-1 H-2-benzopyran-6-carboxamide (PNU109,291; <5.0). Similarly, affinities were established for 13 chemically diverse antagonists, including N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR125,743; p K i, 9.1), (−)-cyanopindolol (9.0), (−)-tertatolol (8.2), N-(4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiozol-3-yl)biphenyl-4-carboxamide (GR127,935; 8.2), N-[3-(1,4-benzodioxan-5-yl)piperidin-4-yl] N-(indan-2yl)amine (S18127; 7.9), metergoline (7.8), (−)-pindolol (7.6), 1′-methyl-5-[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-biphenyl-4-ylcarbonyl]-2,3,6,7-tetrahydro-5 H-spiro[furo[2,3- f]indole-3,4′-piperidine] (SB224,289; 7.5) and ketanserin (<5.0). These rank orders of affinity correspond to the binding profile of 5-HT 1B rather than 5-HT 1D receptors. The low affinities of L775,066 and PNU109,291 versus L694,247 should be noted, as well as the low affinity of ketanserin as compared to SB224,289. Finally, in line with species differences, the affinities of several ligands including CP93,129, RU24,969, (−)-pindolol and (−)-propanolol in rat 5-HT 1B sites were markedly different to guinea pig 5-HT 1B sites labelled with [ 3H]GR125,743. In conclusion, [ 3H]GR125,743 is an appropriate tool for the radiolabelling of native, rat 5-HT 1B receptors and permitted determination of the affinities of an extensive series of ligands at these sites.

Characterization of a Ligand-attenuated Binding Site on Glycoprotein IIb/IIIa

We describe an epitope on the platelet integrin, GPIIb/IIIa, identified by the monoclonal antibody, 4F8, which is attenuated by small-molecule GPIIb/IIIa ligands. 4F8 did not bind to the ligand binding pocket as it did not compete with a radiolabelled antagonist, (3)H-SC-52012. This indicates that the 4F8 epitope behaves as a ligand-attenuated binding site (LABS). Ligand-induced attenuation of 4F8 was an active process as it was prevented by pretreating platelets with cytochalasin D and reduced by prostaglandin E(1) or inhibition of protein kinase C. Disappearance of the epitope was required for full platelet activation as 4F8 prevented platelet aggregation without inhibiting fibrinogen binding. These results suggest a model where disappearance of the 4F8 epitope is a secondary event required for full "outside-in" signaling through GPIIb/IIIa.

NMDA and AMPA glutamate receptor subtypes in the thoracic spinal cord in lean and obese–diabetic ob/ ob mice

Quantitative autoradiography was used to characterise the binding of selective radiolabelled antagonists for the N-methyl- d-aspartate (NMDA) receptor and the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor in the dorsal, intermediate and ventral subregions of the grey matter of the upper thoracic spinal cord in male and female lean and obese–diabetic ( ob/ ob) mice. The density of binding sites for both receptor subtypes was greater in diabetic mice, in all three subregions of the grey matter, than the corresponding subregions in the lean mice. The affinity of the binding site for the NMDA antagonist was significantly higher in obese mice than lean mice, consistent with the presence of two subpopulations of NMDA receptors with different ligand binding affinities in obese mice. The increase in expression of the glutamate receptor subtypes, and altered ligand affinity for the NMDA receptor subtype in the obese mice may be causally involved in the peripheral neuropathies which can accompany diabetes mellitus.

Probing the Immunological Properties of the Extracellular Domains of the Human β1-Adrenoceptor

The human β1-adrenoceptor is an immune target for autoantibodies with functional activity in cardiovascular diseases. Different epitopes on the extracellular domains of the receptor are involved. To study the immunological and pharmacological properties of these epitopes, rabbits were immunized with peptides corresponding to a large domain in the N-terminal part of the receptor and to its first and second extracellular loops. In contrast to the two other peptides, the first extracellular loop did not have immunogenic properties but acted as a hapten. Antibodies affinity-purified with the three synthetic peptides were able to significantly immunoprecipitate the solubilized receptor, confirming that they recognized the target receptor. While antibodies against the N-terminal domain did not inhibit the binding of a radiolabelled antagonist to the receptor, those against the first and second extracellular loop showed non-competitive inhibition. Similarly, only the two latter antibodies exerted a specific agonist-like effect on the receptor, as assessed on neonatal rat cardiomyocytes in culture. Our results are in accordance with those found for human anti-receptor autoantibodies with functional effects. We conclude that not all extracellular epitopes give rise to functional autoantibodies with potential physiopathological relevance in cardiac diseases with an autoimmune component.

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist, in normal and diseased human tissues.

1 We have synthesized a new low molecular weight, non-peptide radioligand, [125I]-PD164333, an analogue of the orally active butenolide antagonists of the endothelin ETA receptor. 2 Analysis of saturation binding assays demonstrated that [125I]-PD164333 bound with high affinity to a single population of receptors (n > or = 3 individuals +/- s.e.mean) in human aorta (KD=0.26+/-0.08 nM; Bmax=8.8+/-3.95 fmol mg(-1) protein), left ventricle from the heart (KD=0.16+/-0.02 nM; Bmax=34.2+/-3.02 fmol mg(-1) protein) and kidney (KD=1.24+/-0.16 nM; Bmax=125.3+/-35.07 fmol mg(-1) protein). In each case Hill slopes were close to unity. 3 In kinetic experiments, the binding of [125I]-PD164333 to ETA receptors in sections of heart was time-dependent and rapid at 23 degrees C. The data were fitted to a one site model, with an association rate constant (K1 of 2.66+/-0.213x10(8) M(-1) min(-1), and a half-time for association of 11 min. The binding was reversible at 23 degrees C: analysis of the data indicated [125I]-PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031+/-0.0004 min(-1), a half-time for dissociation of 216 min and a KD calculated from these kinetic data of 0.01 nM. 4 Unlabelled PD164333 inhibited the binding of [125I]-ET-1 to left ventricle (which expresses both subtypes) in a biphasic manner with a KDETA of 0.99+/-0.32 nM and KDETB of 2.41+/-0.22 microM, giving a selectivity of 2500 fold. ETA-selective ligands competed monophasically for [125I]-PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, KD=3.93+/-0.18 nM; FR139317 KD=3.53+/-0.69 nM. In contrast, the ETB selective agonists, BQ3020 and sarafotoxin S6c (1 microM) did not inhibit binding. 5 In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin-1 (ET-1) concentration-response curve (pA2=8.84) and a slope of unity. 6 In the human brain, autoradiography revealed high levels of [125I]-PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [125I]-PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion. 7 These results show [125I]-PD164333 is a specific, high affinity, reversible non-peptide radioligand for human ETA receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

Characterisation of the binding of [ 3H]MDL 105,519, a radiolabelled antagonist for the N-methyl- d-aspartate-associated glycine site, to pig cortical brain membranes

Binding of [ 3H]MDL 105,519 to glycine sites on N-methyl- d-aspartate (NMDA) receptors of pig cortical brain membranes was evaluated. [ 3H]MDL 105,519 labelled a homogeneous population of binding sites with a K d-value of 3.73±0.43 nM and a B max-value of 3030±330 fmol/mg protein. Clear correlations between the affinity ( K i) to [ 3H]MDL 105,519 labelled sites and the potency (EC 50) to enhance or inhibit non-equilibrium [ 3H]MK-801 binding in the nominal absence of glycine were shown for a variety of glycine site agonists, partial agonists and antagonists. The ratio of K i to EC 50 was >1 for agonists and partial agonists and <1 for antagonists. Various cations as well as glutamate and polyamine site ligands were shown to be able to influence [ 3H]MDL 105,519 binding to pig cortical brain membranes substantially.

Induction of ileum muscarinic cholinoceptor signal transduction pathways by rat interferon-gamma

We have recently shown that rat interferon-gamma (IFNγ) can trigger a cholinergic muscarinic response upon interaction with isolated rat atria, decreasing both the contractile tension and cAMP synthesis, and increasing cGMP production. Because the intestine is rich in cholinergic muscarinic receptors, and IFNγ-producing cells are normal components of the mucosal immune system, we investigated whether IFNγ could also modulate the muscarinic cholinergic activity of the intestine. Our results indicate that IFNγ may interact with ileum cholinergic muscarinic receptors by inhibiting binding of the radiolabelled antagonist quinuclidinyl benzylate ( 3H-QNB), decreasing cAMP formation and increasing cGMP synthesis. These effects are associated with increased mechanical activity of the isolated ileum strips, and can be blunted by pretreatment of the tissue with atropine.

Metabolic tissue characterization in the failing heart by positron emission tomography.

Congestive heart failure represents an important clinical issue which can benefit from newer imaging approaches. Use of radiolabelled pharmaceuticals allow for functional tissue characterization in patients with impaired left ventricular function. Assessment of regional myocardial perfusion and metabolism with either SPECT or PET methods provide accurate detection of tissue viability. Several studies have indicated the utility of these imaging approaches for improved selection of patients for revascularization. The combination of functional measurements with echocardiography and metabolic tissue characterization with PET allows for the non-invasive measurement of cardiac efficiency. The effect of various therapeutic interventions can be objectively assessed by a work-metabolic index. Finally, autonomic innervation of the heart plays an important role in the modulation of the haemodynamic performance of the failing heart. New imaging approaches using radiolabelled catecholamine analogues or receptor antagonists have been used to identify the integrity of myocardiac pre- and post-synaptic sympathetic innervation. Imaging results in patients with dilated cardiomyopathy not only demonstrated a down-regulation of beta-receptor density, but also an abnormal pre-synaptic neuronal function, which may result into impaired regulation of extraneuronal catecholamine concentration. Thus, advanced scintigraphic imaging approaches may improve our diagnostic and prognostic workup in patients with congestive heart failure and may provide objective endpoints for the assessment of therapy.

High affinity [ 3H]formoterol binding sites in lung: characterization and autoradiographic mapping

Agonist binding to the β 2-adrenoceptors and its mapping were studied using the newly developed radioligand [ 3H]formoterol. The results of [ 3H]formoterol saturation binding and formoterol inhibition of [ 3H]formoterol binding were consistent with binding to a single class of receptors ( K d = 1.34 ± 0.15 nM, B max = 154.9 ± 8.0 fmol/mg protein in guinea pig lung membranes, n = 8; K d = 1.05 ± 0.17 nM, B max = 67.8 ± 8.1 fmol/mg protein in human lung membranes, n = 5) and competition assays with other agonists and antagonists disclosed only a single class of site. The nonhydrolyzable GTP analogue GTPγS caused a reduction in both K d and B max, indicating that the receptors labelled by [ 3H]formoterol are coupled to a guanine nucleotide binding regulatory protein. Receptor mapping of [ 3H]formoterol binding sites shows that β 2-adrenoceptors were widely distributed in both guinea pig and human lung, with dense labelling over airway epithelium and uniformly over alveolar walls, and sparse labelling of airway and vascular smooth muscle. In addition, submucosal glands were also sparsely labelled in human bronchus. The distribution of β 2-adrenoceptors was similar to the pattern previously described with non-selective radiolabelled antagonists in the presence of selective β 1-adrenoceptor antagonists.

Characterization of [ 3H]meta-chlorophenylbiguanide binding to 5-HT 3 receptors in N1E-115 neuroblastoma cells

The binding characteristics of a radiolabelled 5-HT 3 receptor agonist, [ 3H]meta-chlorophenylbiguanide (mCPBG), were examined in membranes from N1E-115 neuroblastoma cells. Scatchard plots of saturation binding data showed the presence of two populations of binding sites, with K d = 0.03 ± 0.01 nM and 4.4 ± 1.2 nM and B max = 11.9 ± 4.2 and 897.9 ± 184.7 fmol/mg protein respectively. Competition studies with a selection of agonists revealed the pharmacological profile expected for a 5-HT 3 receptor. The rank order of potency for antagonists was granisetron >quipazine >GR65630 >ondansetron > MDL72222, and for agonists was mCPBG >5-HT (5-hydroxytryptamine, serotonin) >2-methyl-5-HT. IC 50 values for 5-HT and 2-methyl-5-HT were lower than those observed using radiolabelled antagonists, and combined with functional experiments, the data suggest that [ 3H]mCPBG may label high affinity desensitized states of the receptor. We conclude that [ 3H]mCPBG labels 5-HT 3 receptors in N1E-115 neuroblastoma cell membranes and may be a useful compound with which to explore 5-HT 3 receptors in other systems.

MONOCLONAL ANTIBODIES TO ARGININE VASOPRESSIN RECEPTOR BIND TO LIVER, KIDNEY AND PITUITARY MEMBRANES

1. A vasopressin binding protein purified from rat liver membranes was used to immunize Balb/c mice and, subsequently, for the screening of hybrids raised in two different cell fusions. 2. Three hybrids were obtained which secreted monoclonal antibodies (MoAb) that bound to the purified solubilized receptor as detected by an enzyme-linked immunosorbent assay technique. All three MoAb immunoprecipitated the purified receptor. 3. In addition, the MoAb bound in a concentration-dependent manner to crude liver, kidney and anterior pituitary membranes, tissues known to contain arginine vasopressin (AVP) receptors but not to cardiac ventricle membranes which lack AVP receptors. 4. However, the binding of [125I]-[d(CH2)5,Sar7]AVP (a specific radiolabelled V1 antagonist) to the membrane-bound receptor was not inhibited by these antibodies. 5. These results suggest that MoAb recognize epitopes which are common to rat liver, kidney and anterior pituitary membranes but are not at the ligand binding site.

Disrupted β1-adrenoceptor—G protein coupling in the temporal cortex of patients with Alzheimer's disease

The efficacy of β 1-adrenoceptor—G protein coupling was studied in postmortem temporal cortex synaptic membranes from a series of control and Alzheimer's disease subjects. For the control cases, the non-hydrolysable GTP analogue 5′-guanylylimidodiphosphate (Gpp[NH]p) gave a significant reduction in the affinity of the agonist isoprenaline to displace binding of the radiolabelled antagonist (±)-4-(3- t-butylamino-2-hydroxypropoxy)-[5,7- 3H]benzimidazol-2-one ([ 3H]CGP-12177). This effect was attributed to the conversion of high agonist-affinity sites to a lower-affinity state and was not found for the Alzheimer's disease cases. These data indicate that a disruption of β 1-adrenoceptor—G protein coupling occurs in the temporal cortex of Alzheimer's disease patients.

Cholinergic response of isolated rat atria to recombinant rat interferon-γ

Addition of recombinant rat interferon-γ (IFN-γ) to beating rat atria decreased the contractile strength in a dose-dependent manner. The effect was specific of IFN-γ since it was abrogated by monoclonal anti-rat IFN-γ. It required the activation of the cholinergic system of the heart as inhibition of both nicotinic (10 −7 M hexametonium) and muscarinic cholinoceptors (10 −7 M atropine) prevented the reaction. Hemicholinium (2×10 −5 M) and tetrodotoxin (5×10 −7 M) also reduced the response. Likewise, IFN-γ potentiated the action of the muscarinic agonist carbachol. IFN-γ simulated the biological effect of cholinergic agonists because: (a) it increased cGMP formation; (b) it decreased cAMP formation; and (c) it reduced heart contractility at doses that can be considered physiologic. IFN-γ also modified the muscarinic receptor by interfering with the binding of the radiolabelled antagonist quinuclidinyl benzilate ([ 3H]QNB). It is suggested that IFN-γ binding to IFN-γ receptors in the heart may lead to a cholinergic response by interaction of both receptor systems on the surface of atrial cells.

Antilaminin IgG binds and interacts with cardiac cholinergic receptors

Antilaminin IgG bound to cholinergic muscarinic receptors of normal mice heart and simulated the biological effect of a cholinergic agonist. Antilaminin IgG interfered with the binding of the radiolabelled muscarinic antagonist, (-)-[3H]quinuclidinyl benzilate, in a noncompetitive fashion. The interaction of antilaminin IgG with the muscarinic cholinergic receptor increased production of cGMP and decreased production of cAMP. Antilaminin IgG also decreased the contractile tension of mouse atria. Both the mechanical and enzymatic effect of antilaminin IgG required the activation of the muscarinic cholinergic system because they were blunted by atropine and mimicked by acetylcholine.

Two distinct, divalent cation-sensitive, antagonist binding states of heart muscarinic receptors: Differential modulation by guanine nucleotide

1. 1. The binding of [ 3H]quinuclidinylbenzilate (QNB), a muscarinic antagonist, to cardiac muscarinic receptors was investigated in two subcellular fractions (washed particles and microsomes) isolated from rat heart atria and ventricles. 2. 2. 5′-guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM), increased (2-3-fold) the binding to washed particles, but not to microsomes, whereas Mg 2+ (1–20 mM) increased (up to 5-fold) the binding to microsomes, but not to washed particles. Gpp(NH)p modestly increased the affinity while Mg 2+ decreased the affinity towards the radiolabelled antagonist. 3. 3. Treatment with N-ethylmaleimide (NEM, 2 mM) increased the antagonist binding to either fraction. The stimulatory effect of Gpp(NH)p was not evident while that of Mg 2+ survived in the NEM-treated fractions. 4. 4. The treatment of fractions with divalent cations chelators (EDTA, EGTA; 10 mM) augmented the stimulatory effect of Mg 2+ on [ 3H]QNB binding to microsomes while that of Gpp(NH)p on the washed particle [ 3H]QNB binding was decreased. Such treatment further revealed an inhibitory action (about 40%) of Mg 2+ on the washed particle binding.

Three‐dimensional distribution of 3H‐Naloxone binding to opiate receptors in the human fetal and infant brainstem

Despite the putative role of opioids in disorders of the developing human brainstem, little is known about the distribution and ontogeny of opioid-specific perikarya, fibers, terminals, and/or receptors in human fetuses and infants. This study provides baseline information about the quantitative distribution of opiate receptors in the human fetal and infant brainstem. Brainstem sections were analyzed from three fetuses, 19-21 weeks gestation, and seven infants, 45-68 postconceptional weeks, in whom the postmortem interval was less than or equal to 12 hours. Opiate receptors were localized by autoradiographic methods with the radiolabelled antagonist 3H-naloxone. Computer-based methods permitted quantitation of 3H-naloxone binding in specific nuclei, as well as three-dimensional reconstructions of binding patterns. High 3H-naloxone binding corresponds primarily to sensory and limbic nuclei, and to nuclei whose functions are known to be influenced by opioids, e.g., trigeminal nucleus (pain), nucleus tractus solitarii and nucleus parabrachialis medialis (cardio-respiration), and locus coeruleus (arousal). The regional distribution of opiate receptors as determined by 3H-naloxone binding is similar in human infants to that reported in human adults and animals and corresponds most closely to that of mu receptors. We found, however, that opiate receptor binding is high in the fetal and infant inferior olive, in comparison to low binding reported in this site in adult humans, primates, and rodents. In addition, opiate receptors are sparse in the fetal and infant substantia nigra, as in reports of the adult human substantia nigra, compared to moderate densities reported in primates and rodents. By midgestation, the regional distribution of 3H-naloxone binding in human fetuses is similar, but not identical, to that in infants. Highest 3H-naloxone binding occurs in the inferior olive in fetuses at midgestation, compared to the interpeduncular nucleus in infants. Tritiated naloxone binding quantitatively decreases in virtually all nuclei sampled over the last trimester, but not to the same degree. The most substantial binding decrease (two- to fourfold) occurs in the inferior olive and may reflect programmed regressive events, e.g., neuronal loss, during its development. Definitive developmental trends in 3H-naloxone binding are not observed in the postnatal period studied. The heterogeneous distribution of opiate binding in individual brainstem nuclei underscores the need for volumetric sampling in quantitative studies.

Influence of the molecular structure of N 6-(ω-aminoalkyl)adenosines on adenosine receptor affinity and intrinsic activity

The affinities of a series of N 6-(ω-aminoalkyl)adenosines as probes for A 1 and A 2 adenosine receptors were determined in various radioligand binding assays and the intrinsic activities were measured in adenylate cyclase assays. Clear species differences were noticed for A 1 receptor affinity of these adenosine receptor agonists, the compounds being more active in calf than in rat brain tissue. The affinity profile within the series was, however, rather similar in both membrane preparations, with N 6-9-aminononyladenosine displaying highest affinity. The A 2 receptor affinities were comparable to values measured for the A 1 receptor in its low affinity state, as assessed with a radiolabelled antagonist in the presence of 1 mM GTP. Calculation of the intrinsic activities of the adenosine analogues from their modulating action on adenylate cyclase showed almost all the compounds to be equally effective to (−)-N 6-(R-phenylisopropyl) adenosine, on either A 1 or A 2 adenosine receptors. N 6-3-Aminopropyl- and N 6-12-aminododecyladenosine, however, proved to be partial agonists, the first on A 1 and the second on A 2 adenosine receptors. The data are used as the basis for a discussion of adenosine receptor subtype selectivity and intrinsic activity in general.