Radioligand Binding Assay Techniques Research Articles

Quantifying Receptor Properties: the Tissue Segment Binding Method – a Powerful Tool for the Pharmacome Analysis of Native Receptors

The radioligand binding assay technique is an extremely powerful tool for studying receptors. It allows an analysis of the interactions of hormones, neurotransmitters, and related drugs with their receptors. Most of the binding assays have widely been applied to crude membrane fractions prepared from many tissues, but in the conventional method, there are some limitations such as a yield loss of receptor-bearing membranes and a change in receptor environment upon homogenization and fractionation. Recently, in order to overcome these problems, a binding assay has been developed using intact tissue segments. This article presents a brief overview of the tissue segment binding assay that has been developed mainly in our department. Practical guidelines for setting up this new assay are presented, including segment preparation, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. The unique advantages and disadvantages of the tissue segment binding method are discussed in comparison with those of conventional membrane binding methods. We suggest that the tissue segment binding method is a powerful tool for detecting the native properties of receptors occurring in tissues and cells without altering their environment.

PERANAN BIOLOGI MOLEKULER DAN HTS (HIGH THROUGHPUT SCREENING) DALAM PENGEMBANGAN OBAT SINTETIK BARU

Recently, the discovery of new drugs uses the new concept by modern techniques instead ofthe convenstional techniques. In the development of scientific knowledge, the role of molecularbiology and the modern techniques in the investigations and discovery new drug becomes theimportant things. Many methods and modern techniques use in the discovery of new drugs, i.e,genetic enginering, DNA recombinant, radioligand binding assay technique, HTS techniques (HighThroughput Screening), and mass ligand screening. In this review article, its focus in the role ofmolecular biology and HTS techniques (High Throughput Screening) which accelerated thediscovery of new drugs. The role of molecular biology will be the important things on thecharacteristic of structure of protein target as well as receptor that interact with the drug molecule orligand. By the modelling binding receptor technique, its will be known the conformation of bindingreceptor and ligand in the three dimension. In addition, by the quantum mechanics, molecularmechanics, and molecular dynamics will be identified the active sites, the consuming energy, andthe conformation of the structure of molecule drug as well as the receptor when its interactbiochemicaly. In other hand, the investigation of active compounds from natural resources stillconducted. The investigation of active compound from natural resources, at present use the HTStechniques (High Throughput Screening) could screen thousand extracts combine with HumanGenom Project data in this technique. By the HTS techniques (High Throughput Screening) will beperformed screening extract from the natural resources rapidly. Active compounds resulted fromthis technique, subsequently identified and characterized the structure of molecules and synthesisinstead of natural resources. Finally the compounds could be synthesized in the large scale.Themolecular biology and HTS technique will play the big roles on the discovery of new drugs. Themolecular bology techniques related the protein target and receptor have been changed the newparadigm in the design of new molecule drugs by rationally, wheares the HTS technique willaccelerated sceening active compounds from natural resources.Key words: molecular biology, HTS (High Throughput Screening), new drugs

Postnatal development of dopamine receptor expression in rat peripheral blood lymphocytes

Postnatal development in the expression of dopamine D1-like and D2-like receptors was investigated in peripheral blood lymphocytes of male Wistar rats aged 1, 3, 4, 8, 12 and 16 weeks of age by radioligand binding assay techniques. Sample of frontal cortex, striatum and hippocampus were also investigated as reference tissues. The dopamine D1-like receptor antagonist [ 3H]SCH 23390 and the dopamine D2-like receptor agonist [ 3H]7-OH-DPAT were used as radioligands. The affinity ( K d) of [ 3H]SCH 23390 or of [ 3H]7-OH-DPAT binding was unchanged in lymphocytes of rats of different age groups. The density ( B max) of [ 3H]SCH 23390 binding sites increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 8th week of age, and then increased slightly at 12 and 16 weeks of age. The B max value of [ 3H]7-OH-DPAT binding to lymphocytes increased from the 1st to the 3rd week of age, remained constant from the 3rd to the 4th week, increased again until the 12th week and then plateaued. Dopamine D1-like and D2-like receptor maturation in frontal cortex, hippocampus and striatum revealed an increased receptor density until the 4th week of age and a relative stabilization of receptor density values between the 4th to the 12th week depending on the area considered. Comparatively postnatal maturation of lymphocyte dopamine D1-like receptors displayed a pattern different from that of brain areas investigated, whereas maturation of D2-like receptors displayed a pattern similar to that of striatum. The quantitative and/or qualitative dissimilarities between development of lymphocyte and brain dopamine receptors suggest that from a developmental point of view lymphocyte dopamine receptors probably cannot be considered as a marker of homologous brain receptors.

OCCUPANCY BY ORAL ADMINISTRATION OF NICARDIPINE OF L-TYPE CALCIUM CHANNELS IN RAT BRAIN

The occupancy of L-type Ca2+ channels by treatment with an oral dose of the dihydropyridine-type Ca2+ antagonist nicardipine (sustained-release formulation) was evaluated in membrane preparations of rat frontal cortex and hippocampus using a radioligand binding assay technique, with [3H]-nicardipine as a ligand. Three hours after nicardipine administration, specific binding was decreased by about 15–20%, both in the frontal cortex and hippocampus. This indicates that oral nicardipine occupied approximately 15–20% of L-type Ca2+ channels. A progressive occupancy of Ca2+ channels was observed between six and 12 h after nicardipine administration. Twelve hours after drug administration, approximately 65–70% of Ca2+ channels were occupied.These findings indicate that oral treatment with 3 mg/kg of nicardipine (sustained-release formulation) occupies L-type Ca2+ channels in rat brain by more than 40% from the 6th to the 24th h after drug administration. This suggests that an oral dose of nicardipine (sustained-release formulation) induces a significant occupancy of L-type Ca2+ channels in rat frontal cortex and hippocampus for about one day. The possible clinico-therapeutic relevance of this observation is discussed.

Reduced density of dopamine D2-like receptors on peripheral blood lymphocytes in Alzheimer's disease

Clinical and pathological evidence points to an involvement of dopamine in Alzheimer's disease (AD). The present study was designed to assay dopamine D1-like and D2-like receptors on peripheral blood lymphocytes (PBL) in 20 patients with AD and in 25 healthy controls by radioligand binding assay techniques with [ 3H][R]-(+)-(−)chloro-2,3,4,5 tetrahydro-5-phenyl-1H-3-benzazepin-al-hemimaleate (SCH 23390) and [ 3H]7-hydroxy- N, N-di- n-propyl-2-aminotetraline (7OH-DPAT) as radioligands. The density of dopamine D1-like receptors and the affinity of [ 3H]SCH 23390 and [ 3H]7OH-DPAT binding to PBL were similar in both groups investigated. AD patients revealed a lower density of dopamine D2-like receptors on PBL than controls ( P=0.0016). The pharmacological profile of [ 3H]SCH 23390 and [ 3H]7OH-DPAT binding to PBL was consistent with the labeling of dopamine D5 and D3 receptor subtypes, respectively. The reduced density of dopamine D2-like receptors on PBL is consistent with the observation of changes in the expression of D2-like receptors in dopaminergic brain areas in AD. Our findings support the hypothesis of an involvement of dopamine in AD, even in those patients with no evidence of Parkinsonism, behavioral abnormalities or psychosis.

Migraine patients show an increased density of dopamine D3 and D4 receptors on lymphocytes.

Recent studies have revealed peculiar functional and genetic features of dopamine receptors in migraine. As peripheral blood lymphocytes (PBL) may represent a tool for peripheral detection of neuroreceptors, we compared the expression of dopamine D3 (DRD3) and D4 (DRD4) receptors on PBL in migraine patients and in healthy controls using radioligand binding assay techniques in the presence of antidopamine D2-like receptor antibodies. The dopamine D2-like receptor agonist [3H]7-OH-DPAT was used as a radioligand. An increased density of both DRD3 (P=0.0006) and DRD4 (P=0.002) on PBL was observed in migraineurs compared with controls. This up-regulation might reflect central and/or peripheral dopamine receptor hypersensitivity due to hypofunction of the dopaminergic system. These findings support the view that dopamine D2-like receptors are involved in the determination of the so-called migraine trait, which may help to elucidate several clinical features of the disease.

Dopamine D2-like receptors on human peripheral blood lymphocytes: a radioligand binding assay and immunocytochemical study.

1. Peripheral blood lymphocytes express dopamine D1-like and D2-like receptors which were investigated using radioligand binding assay and molecular biology techniques. Analysis of dopamine D2-like receptors expressed by human peripheral blood lymphocytes with radioligand binding assay may offer a rapid technique for assessing receptor changes in disorders characterized by involvement of the dopaminergic system. However, the suitability of radioligand binding assay techniques to measure dopamine D2-like receptors is questioned. 2. In view of the discrepancy between data of dopamine D2-like receptor determination with molecular biology and radioligand binding assay techniques, we have assayed dopamine D2-like receptors expressed by human peripheral blood lymphocytes using as radioligands the dopamine receptor agonist 7-[3H]-hydroxy-N,N-di-n-propyl-2-aminotetraline ([3H]-7-OH-DPAT) and two antagonists ([3H]-spiperone and [3H]-nemonapride). 3. Analysis of saturation curves revealed a concentration-dependent binding of all compounds to human peripheral blood lymphocytes. Dissociation constant (Kd) values averaged between 0.15 and 0.40 nM for different radioligands. The maximum density of binding sites (Bmax) was low, ranging from 4.15 +/- 0.05 fmol/10(6) cells with [3H]-spiperone and 8.66 +/- 0.04 fmol/10(6) cells with [3H]-7-OH-DPAT. 4. Displacement curves of [3H]-7-OH-DPAT, [3H]-spiperone and [3H]-nemonapride binding to human peripheral blood lymphocytes revealed, using radioligand concentrations giving the highest specific:non-specific binding ratio, a pharmacological profile consistent with the labelling of dopamine D2-like receptors. The use of higher radioligand concentrations resulted in a poorly displaceable and characterizable binding. 5. Detection of dopamine D2, D3 and D4 receptor immunoreactivity in cytospin centrifuged peripheral blood lymphocytes revealed dopamine D3 and D4 but not D2 receptor immunostaining. 6. The above findings indicate in agreement with molecular biology studies, that dopamine D2-like receptors expressed by human peripheral blood lymphocytes belong to the D3 and D4 receptor subtypes. These receptors are detectable using either dopamine D2-like receptor agonists and antagonists as radioligands if controlled experimental conditions are followed. The standardisation of immunocytochemical techniques for detecting human peripheral blood lymphocyte dopamine receptors may contribute to clarify their role in lymphocyte function or as a peripheral marker of the status of the dopaminergic system.

Evaluation of an agonist index: affinity ratio for compounds active on muscarinic cholinergic M2 receptors.

A protocol for predicting full agonist, partial agonist, and antagonist profiles of compounds with M2 muscarinic cholinergic receptor activity was developed using radioligand binding assay techniques with [3H]-N-methyl scopolamine (NMS) and [3H]-Oxotremorine-M (Oxo-M) as radioligands. Full muscarinic cholinergic receptor agonists such as muscarine and oxotremorine-M expressed a high agonist index (> 3000 for M1 muscarinic cholinergic receptors and > 900 for M2 muscarinic cholinergic receptor), whereas muscarinic receptor antagonists (selective or non-selective) for different receptor subtypes gave a low (0.5-10) agonist index. Functional studies performed on preparations of guinea-pig ileum and heart were consistent with radioligand binding assay experiments. The above results suggest that similarly as already established for the M1 muscarinic cholinergic receptor subtype, evaluation of the [3H]-NMS/[3H]-Oxo-M ratio may provide useful information on the profile of compounds acting at the M2 muscarinic cholinergic receptor subtype. The availability of simple and predictive techniques for the characterization of muscarinic M2 cholinergic receptor agonists, may help the identification of new compounds in therapeutic areas in which stimulation or inhibition of this receptor is desirable.

Alpha1-adrenergic receptor subtypes in human peripheral blood lymphocytes.

We investigated the expression of alpha1-adrenergic receptor subtypes in intact human peripheral blood lymphocytes using reverse transcription-polymerase chain reaction (RT-PCR) and radioligand binding assay techniques combined with antibodies against the three subtypes of alpha1-adrenergic receptors (alpha1A, alpha1B, and alpha1D). RT-PCR amplified in peripheral blood lymphocytes a 348-bp alpha1A-adrenergic receptor fragment, a 689-bp alpha1B-adrenergic receptor fragment, and a 540-bp alpha1D-adrenergic receptor fragment. Radioligand binding assay with [3H]prazosin as radioligand revealed a high-affinity binding with a dissociation constant value of 0. 65+/-0.05 nmol/L and a maximum density of binding sites of 175. 3+/-20.5 fmol/10(6) cells. The pharmacological profile of [3H]prazosin binding to human peripheral blood lymphocytes was consistent with the labeling of alpha1-adrenergic receptors. Antibodies against alpha1A-, alpha1B-, and alpha1D-receptor subtypes decreased [3H]prazosin binding to a different extent. This indicates that human peripheral blood lymphocytes express the three alpha1-adrenergic receptor subtypes. Of the three different alpha1-adrenergic receptor subtypes, the alpha1B is the most represented and the alpha1D, the least. Future studies should clarify the functional relevance of alpha1-adrenergic receptors expressed by peripheral blood lymphocytes. The identification of these sites may represent a step for evaluating whether they represent a marker of alpha1-adrenergic receptors in cardiovascular disorders or for assessing responses to drug treatment on these receptors.

Ca 2+ channels of the L-type in peripheral blood lymphocytes of essential hypertensives

Ca 2 + channels of the L-type were assayed in human peripheral blood lymphocytes of normotensive control subjects and of essential hypertensives using radioligand binding assay techniques. The dihydropyridine Ca 2 + channel blocker [ 3H]( +)-PN 200-110 [isopropyl1-4-(2,1,3-benzoxadiazol-4-yl)1,4-dihydro-5-methoxycarbonyl-2,6-dimethyl-3-pyridine carboxylate] was used as a ligand. [ 3H]( +)-PN 200 110 was bound specifically to human peripheral blood lymphocytes in a manner consistent with the labeling of Ca 2 + channels of the L-type. No significant differences in the dissociation constant (K d), in the maximum density of binding sites (B max) or in the pharmacological profile of [ 3H]( +)-PN 200 110 binding were found between normotensive subjects and different degree essential hypertensives. Analysis of the intralymphocytic free Ca 2 + concentration did not reveal differences between normotensive subjects and essential hypertensives. Although hypertension is associated with altered membrane handling of Ca 2 + , no changes in the expression of peripheral blood lymphocyte Ca 2 + channels of the L-type or in intralymphocytic Ca 2 + concentrations were found in essential hypertensives. Human peripheral blood lymphocytes therefore cannot represent a peripheral marker of altered Ca 2 + handling in hypertension.

Labeling of dopamine D 3 and D 4 receptor subtypes in human peripheral blood lymphocytes with [ [formula omitted]]7-OH-DPAT: a combined radioligand binding assay and immunochemical study

Molecular biology studies have demonstrated that human peripheral blood lymphocytes express dopamine D 2-like receptors belonging to the D 3 and D 4 receptor subtypes, whereas the characterization of these receptors using radioligand binding assay techniques provided conflicting results. The preferential dopamine D 3 receptor agonist [ 3 H ]7-hydroxy- N, N-di- n-propyl-2-aminotetralin ([ 3 H ]7-OH-DPAT) was used recently for labeling lymphocyte dopamine D 3 receptor. However, the selectivity of this compound for the D 3 receptor was questioned. In this study we have investigated human peripheral blood lymphocyte dopamine receptor subtypes labeled by [ 3 H ]7-OH-DPAT using a conventional radioligand binding assay technique and antibodies against dopamine D 2-like receptor subtypes. [ 3 H ]7-OH-DPAT was specifically bound to intact human peripheral blood lymphocytes with a dissociation constant ( K d) value of 0.32+0.03 nM and a maximum density of binding sites ( B max) of 18.2+0.8 fmol/2×10 6 cells. [ 3 H ]7-OH-DPAT binding was unaffected by antibodies against dopamine D 2 and D 2S receptors. Anti-dopamine D 3 and D 4 receptor antibodies reduced [ 3 H ]7-OH-DPAT binding by about 53% and 32% respectively. Combination of anti D 3 and D 4 receptor antibodies reduced remarkably [ 3 H ]7-OH-DPAT binding. The above results suggest that the dopamine receptor agonist [ 3 H ]7-OH-DPAT labels dopamine D 3 and D 4 receptor subtypes in human peripheral blood lymphocytes. The use of antibodies raised against dopamine receptor subtypes in combination with radioligand binding assay may contribute to define receptor subtypes expressed by human peripheral blood lymphocytes in health and disease.

Peripheral blood lymphocyte muscarinic cholinergic receptors in airway hyperresponsiveness: a marker of cholinergic dysfunction?

1. Muscarinic cholinergic receptors were assayed in human peripheral blood lymphocytes of healthy control and airway hyperresponsive subjects using a radioligand binding assay technique and the muscarinic cholinergic receptor antagonist [3H]-quinuclidinyl benzilate (QNB) as a radioligand. Subjects investigated were divided in four different groups based on threshold responses to methacholine inhaled as challenge test. 2. [3H]-QNB was bound to human peripheral blood lymphocytes in a manner consistent with the labelling of muscarinic cholinergic receptors. Dissociation constant (Kd) values of [3H]-QNB binding were similar in the different groups examined, whereas maximum density of binding sites (Bmax) was increased in airway hyperresponsive subjects in comparison with healthy controls. 3. The above findings indicate that the density of muscarinic cholinergic receptors is increased in peripheral blood lymphocytes of airway hyperresponsive subjects. 4. This suggests that airway hyperresponsiveness is associated with cholinergic hyperreactivity and is probably a systemic cholinergic dysfunction since it is accompanied by changes in the density of muscarinic cholinergic receptors expressed by peripheral blood lymphocytes.

Influence of neonatal treatment with the pyrethroid insecticide cypermethrin on the development of dopamine receptors in the rat kidney

The influence of neonatal treatment with the pyrethroid insecticide cypermethrin (( R, S) α-cyano-3-phenoxybenzyl(1 R, S)- cis- trans-3-(2,2-dichloro-vinyl)-2,2-dimethylcyclopropane carboxylate) on postnatal development of renal dopamine receptors was investigated by radioligand binding assay techniques. Treatment with cypermethrin was made on rats from the 10th to the 16th day after birth. Dopamine D 1-and D 2-like receptors were assayed in frozen sections of kidney of 21-, 30-, 60- and 90-day-old rats using as ligands of dopamine D 1- and D 2-like receptors [ 3H]([ R](+)-(chloro-2,3,4,5,-tetrahydro-5-phenyl-1,4,-benzazepin-al hemimaleate) (SCH 23390) and [ 3H]spiperone, respectively. Treatment with cypermethrin was without effect on the affinity ( K d value) or the density ( B max value) of dopamine D 1- and D 2-like receptors of rats of 21 days of age. In older groups, treatment with the compound reduced the affinity and increased the density of dopamine D 1-like receptors, whereas it was without effect on the affinity of dopamine D 2-like receptors and decreased their density. These findings indicate that neonatal treatment with the pyrethroid insecticide cypermethrin induces long-lasting impairment of renal dopamine D 1- and D 2-like receptors, and that kidney is a target of the toxic action of the compound. Renal dopamine receptor changes caused by cypermethrin are consistent with possible alterations of renal tubular function and of sympathetic neuroeffector modulation. The above data suggest also that, different from the adult, neonatal exposure to pyrethroid insecticides may induce toxic effects.

Dopamine D2-like receptors in the kidney of spontaneously hypertensive rats: a radioligand binding assay and light microscope autoradiography study.

1. Dopamine D2-like receptors were investigated in sections of kidney from male spontaneously hypertensive rats (SHRs) at 6 and 14 weeks of age using radioligand binding assay and autoradiographic techniques with [3H]-spiperone as a ligand. 2. Systolic blood pressure values were slightly higher in 6-week-old SHRs in comparison with age-matched normotensive Wistar-Kyoto (WKY) rats and considerably higher in 14-week-old SHRs in comparison with the other groups investigated. Renal dopamine levels were higher in SHRs aged 6 and 14 weeks in comparison with age-matched WKY rats. Noradrenaline concentrations were similar in 6-week-old SHRs and normotensive WKY rats, and increased slightly in SHRs aged 14 weeks. 3. The density of [3H]-spiperone binding sites was similar in SHRs and WKY rats at 6 weeks of age, and decreased in SHRs at 14 weeks in comparison with age-matched normotensive animals. Light microscope autoradiography revealed the accumulation of silver grains in the tunica adventitia, in the adventitia-media border of intrarenal arteries and within cortical tubules. A few specific silver grains were also developed in the glomerular tuft. No changes in the density and pattern of silver grains were noticeable between SHRs and WKY rats at 6 weeks of age, whereas a reduction in silver grains largely affecting vascular binding sites was observed at 14 weeks of age. 4. Renal denervation considerably decreased the density of [3H]-spiperone binding sites in sections of rat kidney, with an almost complete loss of vascular binding sites. 5. The above findings indicate the occurrence of a decrease of dopamine D2-like receptors in the kidney of SHRs with the progress of hypertension. Dopamine D2-like receptors which are mainly prejunctional are involved in the modulation of sympathetic neurotransmission in the kidney. The loss of these receptors in SHRs may contribute to the pathophysiology of hypertension.

Effect of the Yang tonifying herbs on myocardial β-adrenoceptors of hypothyroid rabbits

New Zealand White female rabbits were randomly divided into three groups, each contained six rabbits, i.e. thyroidectomized and untreated rabbits (group 1), thyroidectomized rabbits treated by the Yang tonifying herbs (group 2) and sham thyroidectomized rabbits as controls (group 3). The myocardial β-adrenoceptor density ( B max) and affinity ( K d) of each group of rabbits were determined by radioligand binding assay technique on the thirtieth postoperative day and the data were handled by using a computer program of the Woolf plot with weight regression. Moreover, the serum levels of thyroxine ( T 4) and triiodothyronine ( T 3) of each group of rabbits were measured by radioimmunoassay technique and their heart rates (HR) were also recorded on the preoperative and thirtieth postoperative day. The results showed that the B max, T 4, T 3 and HR in group 1 were lower significantly than that in group 3 ( P<0.01–0.001), but the change of K d in group 1 was not significant; the deviation of the indices from the normal value in group 2 was less remarkably than in group 1 other than T 4.

Density and Localization of Calcium Channels of the L-Type in Human Pulmonary Artery

The pharmacological profile and the anatomical localization of Ca2+channels of the L-type were investigated in the human pulmonary artery to identify possible mechanisms involved in the regulation of the pulmonary vascular tone. Analysis was performed on slide-mounted frozen sections of human pulmonary artery using radioligand binding assay techniques associated with light microscope autoradiography. [3H]-Nicardipine was used as ligand. Human renal and right coronary arteries also were used as systemic reference arteries. Binding of [3H]-nicardipine to sections of human pulmonary artery was time-, temperature- and concentration-dependent, saturable and reversible. In the human pulmonary artery, the apparent equilibrium dissociation constant (Kd) was 0.12±0.02 nM and the maximum density of binding sites (Bmax) was 38.15±2.25 fmol/mg tissue. Kdvalues were 0.3±0.01 nM and 0.5±0.02 in the human renal artery and right coronary artery respectively. Bmm values were 248 5 16 fmol/mg tissue and 173±9.5 fmol/mg tissue in the human renal artery and right coronary artery respectively. The pharmacological profile of [3H]-nicardipine binding to sections of human pulmonary artery was consistent with the labeling of Ca2+channels of the L-type. It was similar in the pulmonary artery and in the human renal and right coronary arteries. Light microscope autoradiography revealed a high density of [3H]-nicardipine binding sites within smooth muscle of the tunica media of human pulmonary artery as well as of human renal and right coronary arteries. A lower accumulation of the radioligand occurred in the tunica adventitia. No specific binding was noticeable in the tunica intima. Our data suggest that human pulmonary artery expresses Ca2+channels of the L-type sensitive to dihydropyridines. These sites have similar affinity and lower density than those expressed by systemic arteries. The presence of Ca2+channels of the L-type in human pulmonary artery suggests that their pharmacological manipulation may be considered in the treatment of pulmonary hypertension

Dopamine D3 receptor in peripheral mononuclear cells of essential hypertensives.

Dopamine D3 receptor was studied in peripheral mononuclear cells of high-normal, stage 1, stage 2, and stage 3 essential hypertensives using a radioligand binding assay technique with [3H]-7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT) as a radioligand. A group of de novo Parkinsonian patients was also examined as a reference group of impaired dopaminergic function. [3H]-7-OH-DPAT was bound specifically to human peripheral mononuclear cells in a manner consistent with the labeling of a dopamine D3 receptor. No changes in free dopamine, norepinephrine, epinephrine and aldosterone levels, renin activity, dissociation constant of [3H]-7-OH-DPAT binding, or the pharmacological profile of [3H]-7-OH-DPAT binding were found between normotensive control subjects and essential hypertensives or Parkinsonians. The density of peripheral mononuclear cell [3H]-7-OH-DPAT binding sites increased in essential hypertensives parallel to blood pressure value augmentation. A higher density of [3H]-7-OH-DPAT binding sites was found in Parkinsonians. In these patients, the density of [3H]-7-OH-DPAT binding sites was similar to that observed in high-normal subjects and in stage 1 essential hypertensives. The increased density of peripheral mononuclear cell dopamine D3 receptor in hypertension as well as in Parkinson's disease may represent an upregulation mechanism consequent to impaired dopaminergic function. In view of the difficulty in identifying markers of peripheral dopamine function, analysis of dopamine D3 receptor in peripheral mononuclear cells may help evaluate whether the dopaminergic system is involved in hypertension.

Pharmacological characterisation and autoradiographic localisation of a putative dopamine D 3 receptor in the rat kidney

The pharmacological profile and the microanatomical localisation of a putative dopamine D 3 receptor in the rat renal cortex were investigated using radioligand binding assay and light microscope autoradiography techniques. [ 3 H ]7-hydroxy- N, N-di- n-propyl-2-aminotetraline ([ 3 H ]7-OH-DPAT) was used as a ligand. [ 3 H ]7-OH-DPAT was bound specifically to sections of renal cortex. The binding was time-, temperature-, concentration-dependent, of high affinity and guanine nucleotide-insensitive. The dissociation constant ( K d) value was 0.57±0.02 nM and the maximum density of binding sites ( B max) was 62.4±3.5 fmol/mg tissue. The pharmacological profile of [ 3 H ]-7-OH-DPAT binding to sections of rat renal cortex suggests the labelling of a dopamine D 3 receptor. Light microscope autoradiography revealed the accumulation of the radioligand primarily within cortical tubules and to a lesser extent in the glomerular tuft. In glomeruli, binding sites were found mainly in mesangium and mesangial cells. The demonstration of a putative dopamine D 3 receptor in slide-mounted sections of rat renal cortex suggests that appropriate radioligand binding assay techniques combined with autoradiography, may contribute to characterise peripheral dopamine receptor subtypes.

Dopamine D 2-like receptors in the rat kidney: Effect of denervation

The effects of monolateral denervation induced by renal artery occlusion on dopamine D 2-like receptors were assessed in rat kidney using radioligand binding assay and autoradiographic techniques. [ 3 H ]spiperone was used as a ligand. [ 3 H ]spiperone was bound specifically to sections of control innervated kidneys with a dissociation constant ( K d) value of 0.07±0.003 nM and a maximum density of binding sites ( B max) value of 35.4±0.16 fmol/mg tissue. Light microscope autoradiography showed the accumulation of silver grains both in the arterial tree and in cortical tubules. At the vascular level, [ 3 H ]spiperone binding sites were accumulated primarily in the adventitia and in adventitia-media transitional zone. In cortical tubules, the higher density of [ 3 H ]spiperone binding sites was noticeable in proximal convoluted tubules. A few binding sites were also found in the glomerular tuft. In denervated kidneys, noradrenaline and dopamine levels were reduced by about 89% and 60% respectively in comparison with control innervated kidneys. Denervation reduced the density of [ 3 H ]spiperone binding sites by more than 85%. In denervated kidneys, light microscope autoradiography showed the disappearance of specific vascular binding sites and a remarkable reduction of tubular [ 3 H ]spiperone binding sites. The above results indicate that the largest majority of renal dopamine D 2-like receptors labelled by [ 3 H ]spiperone is prejunctional in location.

Use of [3H]‐clozapine as a ligand of the dopamine D4 receptor subtype in peripheral tissues

1. Molecular biology studies have documented the presence of peripheral dopamine D4 receptors. This site has not been characterized yet with classical radioligand binding assay techniques because of the lack of selective radioligands. 2. The atypical neuroleptic clozapine labelled with tritium ([3H]-clozapine) has been proposed and sold as a radioligand for brain dopamine D4 receptors. However, the selectivity of [3H]-clozapine for D4 receptor subtypes, and its specificity for brain dopamine receptors, have been questioned. 3. In this study dopamine D4 receptors were assayed in peripheral organs known to express them, such as rat atria and kidney, by using a radioligand binding assay technique with [3H]-clozapine as the radioligand. Parallel experiments were performed using Chinese hamster ovary (CHO) cells transfected with the D4 receptor clone (variant D4.2). 4. [3H]-Clozapine was bound to sections of rat atria and kidney. After appropriate blockade of sites other than dopamine receptors to which it can bind (i.e. muscarinic cholinergic, serotonergic and alpha-adrenergic receptors), the radioligand was bound to a site displaying a pharmacological profile similar to that expressed by CHO cells transfected with the D4 receptor. 5. The above findings indicate that with appropriate protocols, [3H]-clozapine may represent a radioligand for peripheral dopamine D4 receptors.